Cells of a transformed Syrian hamster line, BP6T, were synchronized by a period of growth in low serum with a subsequent blockage of the cells at the G1/S boundary by hydroxyurea. This method provided cells with nearly 100% synchrony, although approximately 20% of the cells were non-cycling. The cells were then treated with 10(-5) M 5-bromodeoxyuridine for 1 of 5 1-h periods during the S phase and subsequently irradiated with near-ultraviolet light for 5 min. The BrdU plus irradiation treatment induced 6-thioguanine- and ouabain-resistant mutants while BrdU alone or irradiation alone was not mutagenic. 6-Thioguanine-resistant mutants were induced only during early S phase by BrdU plus irradiation treatment. Ouabain-resistant mutants, however, were induced in a biphasic pattern, during early S phase and also during late S phase. The induction of ouabain-resistant mutants at two distinct periods of S phase suggests the presence of two loci for the gene(s) of Na+/K+ ATPase.