We examined the effects of steroid hormones on the proliferation of transformed mouse Leydig cells (B-1) in serum-free culture condition. Among hormones examined, androgen as well as estrogen enhanced the cell proliferation rate. Hormone binding studies revealed that B-1 cells contained both androgen and estrogen receptors. In addition, androgen-enhanced cell growth was inhibited by antiandrogen, but not by antiestrogen, while estrogen-stimulated cell growth was suppressed by antiestrogen. However, the simultaneous addition of androgen and estrogen did not show an additive effect. Dose-response study on androgen-dependent cell growth revealed that relatively high concentrations (10(-7)-10(-6) M) of dihydrotestosterone were required to obtain the maximum response. This was at least partly explained by the finding that B-1 cells could metabolize dihydrotestosterone into the less active steroids. Finally, B-1 cells were found to grow more rapidly in normal than in castrated male mice. These results clearly indicate that the proliferation of B-1 cells is stimulated by both androgen and estrogen, which utilize the different receptor systems.