We have observed quantitative and qualitative differences in the mutability and mutagen-specificity of various drug-resistance marker loci in Chinese hamster ovary (CHO) cells, which suggest that mammalian gene loci may differ in their relative mutability by a given mutagenic agent. We have used the CHO-AT3-2 multiple-marker mutagenesis assay system to examine the dose-dependent induction and kinetics of expression of mutations at four well-characterized, drugresistance marker loci, after treatment with chemical agents which produce various types of DNA damage. The CHO-AT3-2 subline allows simultaneous quantitation and direct comparison of induced mutation frequencies at the hgprt, om (Na'l K+ATPase), uprt, and tk loci. The agents tested in this study included ethyl methanesulfonate, methyl methanesulfonate, mitomycin C, ICR-191, benzo-[alpyrene, and dimethylnitrosamine. The expression kinetics and optimal expression times for each drug-resistance marker were determined in dose-response experiments in which cells from mutagen-treated populations were plated at 1-2-Abbreviations used: AA, 8-azaadenine; APRT, adenine phosphoribosyltransferase, EC 2.4.2.7; BaP benzo[a]pyrene; BUdR, 5-bromodeoxyridine; CHO, Chinese hamster ovary; CHO-AT3-2, subline of CHO heterozygous for the uprt and rk loci; DFBS, dialyzed fetal bovine serum; DMN, dimethylnitrosamine; EMS, ethyl methanesulfonate; FBS, fetal bovine serum; FUdR, 5-fluorodeoxyuridine; HGPRT, hypoxanthine-guanine phosphoribosyltransferase, EC 2.4.2.8; ICR-191, 2-methoxy-6-chloro-9[3-(2chloroethyl) aminopropylamino] acridine dihydmhloride; a-MEM, alpha modified minimal essential medium; MMC, mitomycin C; MMS, methyl methanesulfonate; Na+/K+ATPase, sodiudpotassium activated, magnesiumdependent, membrane bound ATPase, EC 2.6.1.3; OUA, ouabain; PE, plating efficiency; s, surviving cell fraction; SEM, standard error of the mean; SD, standard deviation; TFT, trifluorothymidine; TK, thymidine kinase, EC 2.7.1.21.