Chinese hamster V79 cells were exposed to ethyl methanesulphonate (EMS) and the incidence of mutant cells resistant to 8-azaguanine (8AZG), 6-thioguanine (6TG) or ouabain (OUA) was determined both by the respreading and the in situ techniques. In the former assay, the mutagen-treated cultures were grown for several days to permit the expression of mutations after which the cells were trypsinized, replated (10(5) cells/100-mm dish), and grown in medium supplemented with a selective agent. In the in situ assay, cultures were left undisturbed between EMS treatment and incubation in the presence of the selective agents. The yield of 8AZG-resistant mutants observed at optimal expression times after EMS treatment was comparable for both techniques; the induced mutation frequency (corrected for spontaneous mutation frequency) was estimated to be 82 x 10(-6) mutations per viable cell per unit dose (mM) of EMS. The frequency of 6TG-resistant mutants equalled 45 and 4 x 10(-6)/mM EMS as determined by the respreading and the in situ assays, respectively. In sharp contrast to that observed with 6TG, the frequency of OUA-resistant mutants scored by the in situ assay (30 x 10(-6)/mM EMS) proved to be an order of magnitude greater than that determined by the respreading assay (3 x 10(-6)/mM EMS). Our data therefore indicate that, when OUA is used for mutant selection, the application of the respreading technique, which has been widely adopted as the standard mammalian mutational assay over the past decade, may result in a marked underestimation of the actual mutation frequency (approximately 10-fold in V79 cells).