The aim of this study was to evaluate the potential effect of melatonin on progesterone production by granulosa cells of the Japanese quail. For in vitro experiments, granulosa cells were isolated from pre-ovulatory follicles (F1-F3) when the F1 follicles were predicted to be either immature or mature (at 3-6 or 18-21 h after oviposition, respectively). Granulosa cells were cultured for 12 h with or without melatonin concentration gradients of 0.0001100,ag/mL, thereby averting luteinizing hormone (LH) stimulation. The concentration of progesterone in culture medium was measured using an enzyme immunoassay. The expression of melatonin receptor subtypes in granulosa cells from F1 follicles was detected by reverse transcription-PCR. The LH receptor (LHCGR) mRNA level in cultured granulosa cells of the F1 follicles was analyzed using quantitative real-time PCR. Six quails were used in each of four groups for in vivo experiments. Each group received intraperitoneal injection of melatonin (0.67 mg/kg body weight) or mock-vehicle at 3 or 18 h after oviposition, respectively. The birds were decapitated to collect serum 3 h later (at 6 or 21 h after oviposition, respectively). The serum progesterone level was also measured using an enzyme immunoassay. We observed that melatonin receptor subtypes (Mel-la, lb, and lc) were expressed in the granulosa cells of the F1 follicles of the Japanese quail. Melatonin suppresses the LHCGR mRNA expression in granulosa cells of F1 follicles but does not affect the basal secretion of progesterone in cultured granulosa cells of the F1 F3 follicles. In addition, melatonin treatment has no influence on the serum progesterone concentration at 6h post-oviposition, but suppresses progesterone level 21 h after oviposition in the Japanese quail.