The in situ detection of γH2AX was recently reported to be a promising biomarker of genotoxicity. In addition, the human HepaRG hepatoma cells appear to be relevant for investigating hepatic genotoxicity since they express most of drug metabolizing enzymes and a wild type p53. The aim of this study was to determine whether the automated in situ detection of γH2AX positive HepaRG cells could be relevant for evaluation of genotoxicity after single or long-term repeated in vitro exposure compared to micronucleus assay. Metabolically competent HepaRG cells were treated daily with environmental contaminants and genotoxicity was evaluated after 1, 7 and 14 days. Using these cells, we confirmed the genotoxicity of aflatoxin B1 and benzo(a)pyrene and demonstrated that dimethylbenzanthracene, fipronil and endosulfan previously found genotoxic with comet or micronucleus assays also induced γH2AX phosphorylation. Furthermore, we showed that fluoranthene and bisphenol A induced γH2AX while no effect had been previously reported in HepG2 cells. In addition, induction of γH2AX was observed with some compounds only after 7 days, highlighting the importance of studying long-term effects of low doses of contaminants. Together, our data demonstrate that automated γH2AX detection in metabolically competent HepaRG cells is a suitable high-through put genotoxicity screening assay