We have investigated conditions necessary to quantify the relationship between exposure to a mutagen, ethyl methanesulfonate (EMS), and the frequency of mutation induction at the hypoxanthine-guanine phosphoribosyl transferase locus in V79 cells. Maximal expression of potential mutants has been achieved by either subculturing at fewer than 5 X 10(5) cells/100-mm dish at 2-day intervals or by daily feeding of cultures. An expression period of 5 days (measure from 1 day after the initiation of treatment with the chemical mutagen) should be allowed, since at least 4 days of expression is required to reach to steady maximum of mutation frequency. It appears that there is no concentration dependence of expression time necessary to reach a plateau of mutation frequency with increasing concentrations of EMS up to 1.6 mg/ml. About 1.25 X 10(5) cells/100-mm dish or fewer should be plated for selection to avoid the loss of mutants which occurs at 1.5 X 10(5) cells/dish, presumably through cross-feeding (metabolic cooperation). The use of 6-thioguanine in hypoxanthine-free medium (supplemented with dialyzed fetal calf serum) appears to be a very stringent condition for selection. Mutation induction by EMS as a function of EMS exposure (EMS concentration X treatment time) increases linearly with concentration up to 12 h. For these treatment periods, the observed mutation frequencies for EMS are directly proportional to mutagen exposure regardless of the duration of the treatment.