In a previous work, pinealectomy was found to depress benzodiazepine (BZP) receptor binding in cerebral cortex membranes of rats killed at noon. In order to assess the effect of pineal removal on diurnal variations of BZP binding site concentration and affinity, groups of intact, pinealectomized, or sham-pinealectomized rats (subjected to surgery 2 wk earlier) were killed at six different time intervals during the 24-h cycle. BZP binding was assessed by Scatchard analysis of 3H-flunitrazepam high-affinity binding to cerebral cortex membranes. In intact and sham-pinealectomized rats, a maximum in BZP receptor concentration was found at midnight. Pinealectomy blunted the nocturnal peak of receptor concentration and caused a significant depression of binding site number at noon. No changes in the affinity of the binding sites for the radioligand were detected as a function of time of day or following surgery. In a dose-response experiment for melatonin ability to restore the depressed BZP receptor concentration of cerebral cortex membranes of pinealectomized rats killed at noon, a minimal effective dose of 25 micrograms/kg body weight was obtained. These results further support a link between pineal activity and brain BZP receptors in rats.