Cholinergic effects on hormone secretion by human granulosa cells (GCs) are not well characterized. The aim of this study was to explore the direct action of acetylcholine and carbachol on progesterone and estradiol secretion of human GCs cultured in serum-free medium. Granulosa cells were obtained from 26 women undergoing in vitro fertilization and embryo transfer. Progesterone and estradiol production was measured in the presence and absence of acetylcholine, carbachol, or atropine using radioimmunoassays; statistical analysis of the data was performed by ANOVA. Acetylcholine significantly stimulated progesterone secretion by GCs in a dose-related manner. Estradiol secretion was also stimulated by acetylcholine, but this effect did not show dose dependency. Carbachol showed a similar stimulatory effect, but to a lower degree; both effects can be blocked by acetylcholine. The results suggest that cholinergic action on steroid production by human GCs is mediated through the muscarinic route, and cholinergic neurotransmission may have a physiological significance in the intra-ovarian regulatory pathways.