The objective of this study was to determine if androgens regulate granulosa cell steroidogenesis at physiological doses found in small bovine follicles. Bovine granulosa cells were cultured under serum-free conditions that permit the induction and maintenance of FSH-dependent estradiol secretion. Increasing androstenedione concentrations from 0.1 to 1 or 10 microM significantly increased estradiol accumulation and cytochrome P450 aromatase (P450arom) mRNA abundance. No increase in progesterone accumulation or abundance of mRNA for P450 side-chain cleavage or 3beta-hydroxysteroid dehydrogenase enzymes was observed. The addition of 0.1, 1, or 10 microM progestins or estrogens had no stimulatory effect on P450arom mRNA levels. An analysis of the 5'-untranslated region of P450arom mRNA transcripts indicated that the majority was derived from Cyp19 ovary-specific promoter 2, with some contribution from promoters 1.1 and 1.5. Transcripts from these three promoters were all significantly increased by androstenedione. Testosterone increased promoter 1.1 and 1.5-derived transcripts, but only promoter 2-derived transcripts at the highest dose tested (100 microM). Dihydrotestosterone (DHT) did not affect Cyp19 expression. Collectively, these data show that androgens may exert specific stimulatory effects on P450arom mRNA concentrations in granulosa cells. Interestingly, different androgens had different effects on Cyp19 promoter usage, suggesting differential regulation of aromatase gene expression in the developing follicle.