The in vitro mammalian cytogenetic tests monitor chromosomal aberrations in cultured mammalian cells to test the mutagenicity of compounds. Although these tests are especially useful for evaluating the potential clastogenic effects of chemicals, false positives associated with excessive toxicity occur frequently. There is a growing demand for mechanism-based assays to confirm positive results from cytogenetic tests. We hypothesized that a toxicogenomic approach that is based on gene expression profiles could be used to investigate mechanisms of genotoxicity. Human lymphoblastoid TK6 cells were treated with each of eight different genotoxins that included six DNA damaging compounds-mitomycin C, methyl methanesulfonate, ethyl methanesulfonate, cisplatin, etoposide, hydroxyurea-and two compounds that do not damage DNA-colchicine and adenine. Cells were exposed to each compound for 4h, and Affymetrix U133A microarrays were then used to comprehensively examine gene expression. A statistical analysis was used to select biomarker candidates, and 103 probes met our statistical criteria. Expression of cyclin-dependent kinase inhibitor 1A (CDKN1A)/p21 was ranked highest for discriminating DNA-damaging compounds. To further characterize the biological significance of alterations in gene expression, functional network analysis was performed with the 103 selected probes. Interestingly, a CDKN1A-centered interactome was identified as the most significant network. Together, these findings indicated that DNA-damaging compounds often induced changes in the expression of a large number of these 103 probes and that upregulation of CDKN1A was a common key feature of DNA damage stimuli. The utility of CDKN1A as a biomarker for assessing the genotoxicity of drug candidates was further evaluated; specifically, quantitative RT-PCR was used to assess the effects of 14 additional compounds-including DNA damaging genotoxins and genotoxins that do not damage DNA and five newly-synthesized drug candidates-on CDKN1A expression. In these assays, DNA damage-positive clastogens were clearly separated from DNA damage-negative compounds based on CDKN1A expression. In conclusion, CDKN1A may be a valuable biomarker for identifying DNA damage-inducing clastogens and as a follow-up assay for mammalian cytogenetic tests.