Putative melatonin binding sites were detected in the membrane fraction of gonadotropin-stimulated human granulosa cells using the melatonin analogue 2-[125I]-iodomelatonin (125I-IML). Saturation studies and Scatchard analysis revealed the presence of a major binding site with a Kd of 99 pM. Guanosine triphosphate shifted the receptor affinity to 380 pM. In competition studies, the rank order of potency of indoles for inhibition of 125I-IML binding at these sites was typical of melatonin receptors: 2-iodomelatonin > melatonin > N-acetylserotonin > 5-methoxytryptamine > serotonin. Culture of cells for 7 days in vitro increased receptor density but not the affinity. These findings strongly suggest that melatonin found in follicular fluid may have a physiological role.