A sensitive and stable genotoxic testing system has been developed based on the induction of a Saccharomyces cerevisiae RNR3-lacZ reporter gene expression in response to a broad range of DNA-damaging agents and agents that interfere with DNA synthesis. All 11 tested known carcinogenic and genotoxic agents, ranging from DNA alkylating agents, oxidative chemicals and radiations, were able to induce RNR3-lacZ expression at a sublethal dose. In particular, a potent colon carcinogen, 1,2-dimethyl hydrazine (SDMH), was not detected as a mutagen by a standard Ames test, but was able to induce RNR3-lacZ expression. In contrast, both non-mutagenic and non-genotoxic chemicals tested were unable to induce RNR3-lacZ expression. We have compared three yeast DNA damage-inducible genes for their sensitivity and inducibility, and found that RNR3 is more sensitive than RNR2 and MAG1. The effects of damage dose, post-treatment incubation time and cell growth stage on RNR3-lacZ expression have also been determined and optimized. In order to create a stable and user-friendly testing system, we integrated the RNR3-lacZ cassette into the yeast genome and demonstrated that its inducibility is indistinguishable from that of plasmid-based studies.