A forward mutation assay based on 5-fluorouracil (FU) resistance has been developed using a strain of Salmonella typhimurium derived from the Ames strain TA100. The sensitivity of the assay benefits from the genetic characteristics present in the standard Ames strain that enhances the response to genotoxic agents. A mutation conferring resistance to 5-fluorouridine was also introduced into the test strain to avoid unwanted toxicity resulting from cross-feeding of 5-fluorouridine between wild-type and FU-resistant (FU(R)) cells during selection with FU. In the mutation assay 1 ml aliquots of exponentially growing bacteria are exposed to the test agent for 2 h in the presence and absence of a rat-liver S-9 metabolizing system. The aliquots are then diluted, allowed to grow for 3 h, and assessed for treatment-related toxicity/inhibition by optical density. The cultures are diluted a second time and grown overnight to permit full recovery from toxicity and to allow expression of the FU(R) phenotype. Samples of the cultures are then plated in 384-well microtiter dishes in the presence of 2 microg/ml FU and the pH-sensitive indicator bromcresol purple. Three days later positive wells containing FU(R) colonies are detected by their yellow color and enumerated. Results were obtained using a variety of 45 compounds to validate the assay. Of the 25 mutagens and 20 non-mutagens tested, the results correlated 100% with those collected using the battery of standard Ames reversion strains, further supporting the use of the FU Assay as an alternative screen to the Ames assay. The use of a single strain exposed in liquid suspension permits assessment of high concentrations of test compound but with a low overall compound requirement (30 mg total). The highly parallel nature of culture handling/dilution and use of standard microtiter plates also offers the possibility of assay automation.