Effects of pinealectomy and melatonin replacement on the timing of the preovulatory luteinizing hormone (LH) surge were examined in female rats housed under a light-dark cycle (lights on 06:30–18:30 h). Animals were pinealectomized or sham-operated 21-28 days before bleeding. They were sequentially bled in the afternoon of proestrus through the indwelling cardiac cannula without anesthesia. Proestrous LH surges were observed in all pinealectomized rats, but the onset times of the LH surge in these animals showed a significantly greater variance than those in sham-operated controls. A single melatonin injection at 18:00 or 21:00, shortly before or after the light-dark transition, on the day before proestrus (diestrus II) reduced the variance in the LH surge onset in pinealectomized rats in a dose-dependent manner. In contrast, melatonin injected at 14:00 or 23:00 on diestrus II or at 04:00 on proestrus was ineffective. These results showed that the pineal gland is involved in controlling the timing of the proestrous LH surge in the rat. The pineal signal may be transmitted by melatonin, a major product of the glan