tert.-Butylhydroquinone (TBHQ) has been reported to be genotoxic in some short-term assays but non-genotoxic in others. We have examined cytotoxicity and genotoxicity of TBHQ, a principal metabolite of the phenolic antioxidant 2(3)-tert.-butyl-4-hydroxyanisole (BHA), in an hepatocyte-mediated assay with V79 Chinese hamster lung cells including both sister-chromatid exchange (SCE) and thioguanine-resistance (TGR) endpoints. The ability of BHA and of TBHQ to elicit a genotoxic response in Saccharomyces cerevisiae strain D7 was also investigated. In V79 cytotoxicity tests, TBHQ without hepatocytes produced a 50% reduction in colony formation at 4.2 micrograms/ml and was lethal to 100% of the cells at concentrations above 5 micrograms/ml. At partially cytotoxic dose levels, (0.17-3.4 micrograms/ml of medium), TBHQ sometimes increased significantly the frequency of SCE. TBHQ also produced sporadic statistically significant increases in the mutation frequency at the HGPRTase (TGR) gene locus when tested alone or with activation by rat or hamster hepatocytes. Mitotic gene conversion and reverse mutation were not induced in strain D7 of Saccharomyces cerevisiae by exposure to BHA or to TBHQ for 4 h at concentrations as high as 200 micrograms/ml for BHA or 500 micrograms/ml for TBHQ, either alone or with activation by rat-liver S9. Incubation of the yeast cells with BHA or TBHQ for 24 h in growth medium without activation also did not induce genotoxic activity. The slight and sporadic response to TBHQ in the V79 test system may indicate weak genotoxicity which is sensitive to slight differences in test conditions. The classification and test strategies adopted for compounds such as TBHQ could have important implications for regulatory decisions and for the validation of short-term tests.