Rickettsia rickettsii is the tick-borne etiologic agent of Rocky Mountain spotted fever. R. rickettsii is the prototypic spotted fever group rickettsia. Several other species, R. conorii, R. siberica, R. japonica, R. akari, and others cause diseases of lesser severity. Still other species in the spotted fever group, R. montana, R. peacockii, R. belli, and R. rhipicephali, are considered avirulent as they have never been associated with human disease nor do they cause overt disease in standard laboratory animals. The typhus group of rickettsia, typified by R. prowazeki, the agent of epidemic typhus, include some of the historically most devastating disease agents known to mankind. The typhus group also includes species of lesser or no virulence potential to humans. R. prowazeki and R. rickettsii are classified as Biodefense Catagory B and C agents, respectively. Rickettsia rickettsii is a member of the spotted fever group rickettsiae and the etiologic agent of Rocky Mountain spotted fever (RMSF). R. rickettsii is a small obligate intracellular Gram-negative organism maintained in its tick host through transovarial transmission. Infection with R. rickettsii occurs through the bite of an infected tick 33. Once the organism gains access to the host it is able to replicate within the host vascular endothelial cells and spread from cell to cell by polymerizing host cell actin. Damage to vascular endothelial cells by R. rickettsii leads to increased vascular permeability and leakage of fluid into the interstices causing the characteristic rash observed in RMSF. Infection with R. rickettsii results in a severe and potentially life threatening disease if not diagnosed and treated properly. While much is known about the progression of disease, the molecular mechanisms involved in the pathogenesis of RMSF are poorly understood. Strains of Rickettsia rickettsii vary dramatically in their virulence in animal model systems and severity of human disease. The obligate intracellular lifestyle of rickettsiae and the lack of tractable genetic systems make it difficult to identify genes involved in virulence. With the completed sequences of multiple rickettsial species, it has become possible to investigate differences between virulent and avirulent strains of rickettsiae through comparative genomics. In efforts to generate isogenic mutant pairs to definitively identify rickettsial virulence determinants, we used the mariner-based transposon mutagenesis system to generate a collection of R. rickettsii mutants. Direct sequencing of purified genomic DNA from each of the clones using primers from within the transposon delineated the precise insertion sites. Transposition sites were randomly distributed throughout the rickettsial genome. Three of the clones contained transposon insertions in intergenic regions while the remaining five clones contained a single insertion in the open reading frames. One transposon insertion site was within Sca2, a member of a family of large autotransporter proteins. The clone in which Sca2 was disrupted (clone 9-7) displayed a unique plaque morphology in which the plaques were visibly smaller relative to the parental strain. The transposon mutants and wild-type rickettsiae replicated at equivalent rates demonstrating that differences in intracellular multiplication could not account for the differences in plaque size. Because plaque size is often correlated with capacity for bacteria to spread to adjacent cells, we used phalloidin staining of infected Vero cell monolayers to investigate whether the Sca2 mutant was able to generate actin comet tails. Long actin tails were readily detected emanating from a single pole of wild-type rickettsia. In contrast, actin comet tails were not observed in cells infected with Sca2 mutant bacteria (clone 9-7) nor was actin visibly recruited to the surface of the microbe. An unrelated transposon mutant clone 9-3 behaved as the parent rickettsiae. Notably, the Sca2 mutant did not appear to spread readily to adjacent cells as did the parent rickettsia or the clone 9-3. The tendency to remain in an infected cell and replicate without spread to neighboring cells is characteristic of R. prowazekii which lacks actin-based motility . Altogether, the data suggest that Sca2 is necessary for rickettsial actin-based motility and cell to cell spread. Compared to the parent strain or a transformant with a different insertion site, the Sca2 mutant displays reduced virulence in a Guinea pig model of infection. Sca2 and actin-based motility therefore appear to be bone fide virulence determinants of spotted fever group rickettsiae.