A potential role of insulin-like growth factor I (IGF-I) in the regulation of steroidogenesis in the rabbit corpus luteum was investigated using a primary culture of luteal cells obtained 3 days after ovulation. Dissociated cells were cultured for 1 day in the presence of medium 199 and 10% fetal bovine serum; thereafter, the cells were cultured in medium 199 containing 0.1% BSA, gentamicin (50 micrograms/ml), and hormones or growth factors, and without serum. IGF-I (human recombinant, 100 ng/ml) was as effective as LH (ovine, 10 ng/ml) in maintaining progesterone accumulation through 4 days of culture. Estradiol (10(-8) M), either alone or in combination with LH or IGF-I failed to stimulate progesterone accumulation, which was not surprising since these cells did not possess estrogen receptors. The stimulation of progesterone by IGF-I was not detectable until 24-36 h after introduction of the growth factor to the cultures, whereas stimulation by LH was observed within 2 h. The steroidogenic effect of IGF-I was not attributable to increased cell number, as DNA values or [3H]thymidine incorporation were unchanged by IGF-I. IGF-I increased functional enzymatic activity, observed as increased progesterone accumulation in the presence of 25-hydroxycholesterol used as exogenous substrate. These data indicate that luteal cells have the capacity to respond to IGF-I, raising the possibility that IGF-I has a role in the regulation of steroidogenesis in the rabbit corpus luteum.