A direct effect of physiological levels of testosterone on skeletal muscle cells in tissue culture is reported. The effect is a 25% stimulation of labeling index by radioautographic analysis of cultures following incubation with [3H]thymidine at the end of a 48-h exposure to 10(-8)M testosterone in 2% gelding serum. This stimulation was observed in primary myoblasts, an established myogenic cell line (Yaffe's L6 cells), and muscle fibroblasts. The effect was specific for testosterone: the labeling index did not change significantly from control values when estradiol, 5beta-pregnanediol, androstenedione, or dihydrotestosterone were added, although small increases were noted in the latter two cases. The effect on the labeling index was localized as a decrease in time spent in the G1 phase of the cell cycle, which decreased about 30% in muscle cells exposed to testosterone. This first report of an effect of testosterone on isolated muscle cells, coupled with the recent description of a receptor for anabolic steroids in muscle cytoplasm, indicates that the effects of male sex hormones result from a direct interaction with muscle rather than from a primary interaction with some other tissue.