The interaction of normal human diploid cells and chemical carcinogens and mutagens was studied in-vitro. Human fibroblast lines were derived from neonatal foreskins. Synchronous and non synchronous fibroblasts were treated with single or multiple doses of 7,12-dimethylbenz(a)anthracene (57976) (DMBA) or 4-aminobiphenyl (92671) in the presence of rat liver S9 mix and assayed for 6- thioguanine resistant mutants. Both DMBA and aminobiphenyl induced dose dependent mutants after either treatment protocol. Multiple treatment of non synchronized cells caused more cell death than did single treatment of synchronized cells. Non synchronized human fibroblasts were treated with multiple exposures of methylmethanesulfonate (66273) (MMS) for up to 72 hours and assayed for thioguanine resistant mutants as before. Exposure to MMS for 24 hours did not induce mutations; however, after 72 hours a dose dependent increase in mutation frequency occurred. Non synchronized human fibroblasts were treated with multiple doses of N-ethyl-N- nitrosourea (759739) (ENU), N,N-bis(2-chloroethyl)-N-nitrosourea (154938) (CENU), cyclophosphamide (50180), or melphalan (148823) with or without metabolic activation with S9 mix and assayed for thioguanine resistant mutants as before. ENU was a potent direct acting mutagen and CENU a weak direct acting mutagen. Melphalan and cyclophosphamide required metabolic activation to express mutagenic activity. Melphalan appeared to be the more potent mutagen. Human fibroblasts were treated with MMS during the G1 plus-S, S-plus G2, or G2 plus G1 phases of the cell cycle. The cultures were assayed for thioguanine resistant mutants as before. MMS induced mutations during the G1 plus-S and S-plus G2 phases, but not the G2 plus G1 phase. The authors conclude that only S-phase cells are sensitive to mutagens and that S-phase cells must be included in mutagenesis experiments.