Chemical mutagens including methyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, iodomethane, and epichlorohydrin have been classified as "chromosomal mutagens" in the L5178Y/thymidine kinase (TK) gene mutation assay by Clive and coworkers [Mutat Res 59:61-108, 1979; and "The Predictive Value of Short-Term Screening Tests in Carcinogenicity Evaluation." Amsterdam: Elsevier/North Holland, pp 103-123, 1980] who observed mutagen-dependent increases in small TK-deficient mutant colonies with detectable damage to the chromosome (11) that carries the TK locus. In this study, we tested these four chemicals for the induction of gene mutations at the ouabain-resistance (ouares) locus of 3.7.2C L5178Y cells to determine if presumptive chromosomal mutagens would go undetected at a gene locus that is unresponsive to chromosomal damage. A final concentration of 375 micrograms/ml ouabain in soft-agar medium selected against the ouabain-sensitive phenotype without loss of the mutagen-induced ouabain-resistant phenotype. Verification of the mutant phenotype was completed for six individual soft-agar ouares colonies derived from mutagen-treated cultures via growth for 10-11 days in nonselective medium followed by retesting for colony formation in selective soft-agar medium. Dose-related reproducible increases in the frequency of ouabain-resistant mutants were observed for 3.7.2C L5178Y cells that had been exposed for 3 hr to 24-46 micrograms/ml epichlorohydrin, 1.9-3.6 micrograms/ml iodomethane, 0.006-0.011 micrograms/ml N-methyl-N'-nitro-N-nitrosoguanidine and and 2.0-5.4 micrograms/ml methyl methanesulfonate. Also, treatments with EMS, which induced sufficient numbers of ouares colonies to permit analysis of colony size distribution, showed the existence of a bimodal size distribution similar to those reported for TK-deficient mutants. This discovery suggests that mutant colony size in this cell line may be independent of specific gene locus effects. We conclude that (1) chemicals that induce a high proportion of chromosomal mutants, as detected at the TK locus in earlier studies, also induce single gene mutations at the ouabain-resistance locus and (2) a bimodal distribution of mutant colony sizes in soft-agar medium after short expression periods may be a distinctive characteristic of the 3.7.2C L5178Y cell line and is not confined to the TK locus.