The in vitro micronucleus (IVM) test is currently used as a screen during the early stages of pharmaceutical development to identify chemicals likely to produce positive outcomes in the in vitro chromosome aberration assay. For several reasons, the assay is being considered as an alternative to the aberration assay, but the current screening protocols are not rigorous enough to fully satisfy concerns about genotoxic safety. This manuscript describes the investigation of several protocol parameters to assist with the development of a regulatory guideline for the IVM test. The parameters investigated are: the effect of cytochalasin B on the outcome of the assay when conducted with continually growing cell lines; the need for an extended exposure in the absence of metabolic activation; and the number of cells to be counted for a valid assay. In addition, two statistical procedures for the analysis of data from the test are described. The results of the investigation indicate that cytochalasin B does not effect the outcome of the test, that the extended exposure treatment is not necessary, that counting 2000 cells is preferable to counting 1000, and that the data can be appropriately analyzed using a trend test.