DNA double-strand breaks (DSBs) and blocked replication forks resulting from bulky adducts and inhibitors of replication activate the DNA damage response (DDR), a signaling pathway marked by phosphorylation of histone 2AX (H2AX). The phosphorylated form, γH2AX, accumulates at the site of the damage and can be visualized as foci by immunocytochemistry. The objective of this study was to assess if γH2AX is a reliable biomarker for genotoxic exposures. To this end, we selected 14 well-known genotoxic compounds and compared them with 10 nongenotoxic chemicals, using CHO-9 cells because they are well characterized as to DNA repair and DDR. We quantified γH2AX foci manually and automatically. In addition, total γH2AX activation was determined by flow cytometry. For all chemicals the cytotoxic dose response was assayed by a metabolic cytotoxicity assay. We show that (1) all genotoxic agents induced γH2AX dose-dependently whereas nongenotoxic agents do not; (2) γH2AX was observed for genotoxicants in the cytotoxic dose range, revealing a correlation between cytotoxicity and γH2AX for genotoxic agents; for nongenotoxic agents cytotoxicity was not related to γH2AX; (3) manual scoring of γH2AX and automated scoring provided comparable results, the automated scoring was faster and investigator independent; (4) data obtained by foci counting and flow cytometry showed a high correlation, suggesting that γH2AX scoring by flow cytometry has the potential for high-throughput analysis. However, the microscopic evaluation can provide additional information as to foci size, distribution, colocalization and background staining; (5) γH2AX foci were colocalized with 53BP1 and Rad51, supporting the notion that they represent true DSBs. Collectively, the automated analysis of γH2AX foci allows for rapid determination of genetic damage in mammalian cells. The data revealed that the induction of γH2AX by genotoxicants is related to loss of viability and support γH2AX as a reliable bio-indicator for pretoxic DNA damage.