The objective of this in vitro study was to determine whether the increase in the augmented phase of the biphasic luteinizing hormone (LH) response to gonadotrophin-releasing hormone (GnRH) and its enhancement by estradiol (E2) were associated with GnRH-stimulated increases in pituitary GnRH receptor concentration. Pituitary glands from 72 h ovariectomized (OVX), OVX + E2, or proestrous rats were perifused continuously with GnRH (12 ng/h). LH release was measured at 10-min intervals, and pituitary GnRH-binding capacity (GnRH-BC) was assessed at 0, 40, 80, 120, and 240 min after addition of GnRH. All treatment groups exhibited a biphasic pattern of LH release; initial (20-70 min) and augmented (120-240 min) mean rates of LH secretion (micrograms/h) were 1.78 and 3.92 (OVX), 6.40 and 16.67 (OVX + E2), and 2.79 and 18.64 (proestrus), respectively. Total LH release was significantly greater in the OVX + E2 and proestrous groups (44.0 and 45.8 micrograms) vs. the OVX group (12.4 micrograms). Throughout the GnRH infusion period, GnRH-BC did not change significantly in any of the treatment groups with the exception of the OVX group in which there was a transient small decrease at 80 min post-GnRH infusion. There were no significant differences between treatment groups in GnRH-BC at any time after infusion of GnRH. These results demonstrate that the acute and augmented phases of GnRH-stimulated LH release and the enhancement of this biphasic response by E2 occurs independent of any increase in GnRH-BC and suggest that these events are mediated by postreceptor mechanisms.