In order to assess the effect of pinealectomy (Px) on the diurnal rhythmicity of gamma-aminobutyric acid (GABA) high affinity binding to cerebral cortex membranes, groups of intact, Px or sham Px rats (subjected to surgery 15 days earlier) were killed at six different time intervals during the 24-hour cycle. GABA binding was estimated by Scatchard analysis of 3H-GABA binding to cerebral cortex membranes prepared from individual brains; only one type of binding site with dissociation constant (KD) about 20-50 nM and site number (Bmax) about 200-500 fmol/mg protein was apparent in the assay conditions employed. In intact and sham Px rats Bmax attained minimal values at night and increased during daylight. Px increased generally Bmax and disrupted its normal diurnal rhythmicity, a peak in Bmax being observed at midnight. A significant decrease of GABA high affinity binding affinity was detected at morning hours in intact rats and at late scotophase and morning hours in Px and sham Px rats. Bmax of GABA high affinity binding in Px rats attained maximal values by 5-10 days after surgery and decreased somewhat 5 days later. Sham Px rats exhibited a transient increase in Bmax up to 10 days after surgery, returning to normal values by the 15th day. Superior cervical ganglionectomy increased binding affinity up to 15 days after surgery without affecting Bmax. The minimal melatonin effective dose to counteract Px-induced increase of GABA high affinity binding was 25 micrograms/kg body weight when given 3 h before sacrifice. Melatonin activity on GABA binding did not depend upon a direct effect on the binding sites, as shown in vitro. These results suggest a link between pineal function, melatonin secretion and GABA receptor activity in rats.