The in vitro micronucleus (MN) assay is used to assess the genotoxic potential of a test substance by measuring the induction of micronuclei in cultured mammalian cells. The flow cytometry-based assay (in vitro MicroFlow^® kit, Litron Laboratories, USA) coupled with a high-throughput sampler permits the analysis of thousands of nuclei over a short period of time. Furthermore, clastogenic or aneugenic mechanistic signatures can be discerned using this high content approach. Using this version of the assay, the genotoxicity of the total particulate matter (TPM) fraction and the saline-soluble portion of the gas-vapour phase (GVP) derived from 3R4F reference cigarettes (University of Kentucky, USA) along with several prototypical genotoxins was evaluated using a study design compliant with OECD guideline No. 487 and recommendations from Litron Laboratories. In order to maximise the possibility of detecting a genotoxic response, three different treatment conditions were deployed in CHO-K1 cells: extended (24 h), short-term without metabolic activation (4 h -S9) and short-term in the presence of a S9 metabolic activation system (4 h +S9). In order to understand cytotoxicity vis-à-vis genotoxicity in the assay, three different cytotoxicity indices were explored, namely relative population doubling (RPD), relative increase in cell counts (RICC) and relative cell counts (RCC) all of which were calculated from absolute cell counts measured by flow cytometry. Known clastogens, e.g. methyl methanesulphonate, and aneugens, e.g. colchicine, were shown to induce marked genotoxicity as well as clear clastogenic and aneugenic mechanistic signatures, while several non-genotoxic cytotoxins, e.g. phthalic anhydride, were confirmed as non-genotoxic in the assay. 3R4F-derived TPM and GVP induced clastogenic responses but there was no detectable evidence of aneugenicity. Cytotoxicity was underestimated by RCC for most of the substances tested, whereas RICC appeared to be the most sensitive index and RPD typically yielded levels of cytotoxicity midway between RCC and RICC. In conclusion, the flow cytometry-based in vitro MN assay is appropriate to evaluate the genotoxic potential of tobacco smoke-derived aerosols.