The γH2AX focus assay, based on phosphorylation of the variant histone protein H2AX, was evaluated as a genotoxicity test in immortalised wild-type mouse embryonic fibroblasts (MEFs) treated for 4h with a panel of reference compounds routinely used in genotoxicity testing. The topoisomerase II poison etoposide (0.006–60μg/ml), the alkylating agent methyl methanesulfonate (1.3–65μg/ml) and the direct DNA-damaging agent bleomycin (0.1–10μg/ml) all produced a positive concentration–response relationship. The non-genotoxic compounds ampicillin (0.035–3500μg/ml) and sodium chloride (0.058–580μg/ml) showed no such response with increased concentrations. The H2AX phosphorylation results were compared with the outcome of two standard in vitro genotoxicity tests, namely the micronucleus and comet assays. Compounds that produced measurable DNA damage in the focus assay generated micronuclei at comparable concentrations. In this study, the focus assay identified genotoxic agents with the same specificity as the comet assay. These results were substantiated when H2AX phosphorylation was analysed using flow cytometry in the murine cell line L5178Y, growing in suspension. The data were in concordance with the manual scoring focus assay. To further this investigation, the γH2AX flow cytometry was compared to the in vitro micronucleus flow cytometry and mouse lymphoma assay using the same cell population after MMS treatment. The median γH2AX value increased significantly above the control at all four MMS concentrations tested. The percentage of micronucleus events in the in vitro micronucleus flow test and the mutation frequency in the mouse lymphoma assay were also significantly increased at each MMS concentration. The current data indicate that H2AX phosphorylation could be used as a biomarker of genotoxicity, which could predict the outcome of in vitro mammalian cell genotoxicity assays.