Gonadotropin secretion is diminished in the presence of hyperprolactinemia, and previous studies have shown that PRL can reduce GnRH secretion and impair LH responses to GnRH. To investigate the mechanisms of the inhibitory effects of PRL on the pituitary, we administered intraarterial pulse injections of GnRH (25 ng/pulse every 30 min) to castrate testosterone-implanted male rats placed in restraint cages. Serum PRL, GnRH receptor (GnRH-R), and LH responses to GnRH were measured at intervals over 72 h. In control animals which received saline pulses, serum PRL was transiently elevated to the range of 100-150 ng/ml during the first 24 h, GnRH-R remained stable (approximately 300 fmol/mg protein) and serum LH was low (less than 10 ng/ml) throughout the 72 h. GnRH pulses in castrate testosterone-implanted animals increased GnRH-R to values (approximately 600 fmol/mg) similar to those in castrate controls (no testosterone implant, saline pulses) through 48 h, but GnRH-R declined to baseline values by 72 h in both groups. Serum LH responses to GnRH pulses were only present at 24 h. Administration of bromocriptine throughout the 72 h to immobilized castrate rats or to castrate testosterone-replaced animals treated with GnRH pulses suppressed serum PRL, and GnRH-R concentrations remained elevated through 72 h. Serum LH responses to GnRH pulses were 5- to 20-fold higher in bromocriptine-treated rats, and responses were present throughout the 72 h of the experiment. Delaying the start of bromocriptine treatment until 36 h (after the spontaneous PRL peak) resulted in reduced GnRH-R and LH responses at 72 h. Similarly, administration of ovine PRL (during the first 48 h) to bromocriptine-treated rats produced low GnRH-R concentrations at 72 h. Thus, the transient elevation of PRL seen in immobilized rats can inhibit the GnRH-stimulated increase in GnRH-R and is associated with reduced LH responses to GnRH. These results indicate that PRL has a direct inhibitory effect on the gonadotrope and suggest that impaired GnRH-R responses to GnRH are one of the mechanisms involved in the diminished gonadotropin secretion seen in hyperprolactinemia.