In order to assess the direct luteotropic effects of steroids, dispersed luteal cells from rats on day 8 or 16 of pregnancy were incubated for 2 h with various concentrations of estradiol (1-1000 ng/ml), testosterone (1-100 ng/ml) or progesterone (1-100 ng/ml). Estradiol, at any concentration, did not cause significant changes in the in vitro accumulation of progesterone, 20 alpha-dihydroprogesterone (20 alpha-OHP) or testosterone by dispersed luteal cells on both days 8 and 16. Addition of testosterone (1-100 ng/ml) increased the accumulation of estradiol on day 8 with estrone synthesized at half to one third the rate of estradiol. On day 16, accumulation of estradiol by dispersed luteal cells was more sensitive to 1 ng/ml testosterone than on day 8 with a concomitant drop in estrone synthesis. Although luteal secretion of estradiol was readily stimulated by testosterone, there were no corresponding changes in the production of progesterone or 20 alpha-OHP. Although the addition of a small amount of progesterone did not cause the anticipated increase in accumulation of 20 alpha-OHP, 100 ng/ml progesterone elicited a slight but significant increase in accumulation of 20 alpha-OHP on day 8. Thus, none of 3 steroids added was found to exert any direct in vitro luteotropic action in the pregnant rat, in acute experiments.