The organization of mitochondrial DNA in maize contributes to both aspects of organismal fitness: survivability and fecundity. Rearrangements in mtDNA are correlated with male sterility and in one case with increased susceptibility to fungal pathogens. Variation in maize mtDNA may be generated by intra- and interchromosomal rearrangements that are maternally transmitted to the next generation. The mitochondrial DNA of maize and other higher plants is very complex. Multiple circular size classes are visualized by electron microscopy of DNA samples, and restriction digests indicate a variety of stoichiometric yields of different fragments. These results may be explained by intramolecular recombination and subsequent amplification of a subset of the base genome. To resolve the organization and replication of maize mtDNA we are constructing a physical map of the genome and propose to determine the distribution of origins of replication. We are constructing a physical map of the mitochondrial genome using cloned Eco RI and Bam HI fragments of normal mitochondrial DNA. When completed the 475kb genomic map of maize mtDNA will be the largest entity with a physical map. We have already determined the organization of the multiple, dispersed 17S and 26S ribosomal RNA genes, and these will be placed on the physical map. A cDNA library of mitochondrial RNA sequences prepared to determine which regions of the genome are transcribed. Utilizing psoralen cross-linking of DNA we will describe the origin(s) of replication for this genome. This study will be complemented by examining the ability of mitochondrial DNA sequences to function as autonomous replicating sequences/origins in plasmids of yeast and E. coli.