Previous reports have demonstrated that long-term continuous administration of gonadotropin-releasing hormone (GnRH) to pituitary cells results in a decreased level of gonadotropin secretion. The present report demonstrates that cultured rat anterior pituitary cells preincubated for 6 h in 10(-9) or 10(-7) M GnRH became refractory to further stimulation by the releasing hormone. Cells required 2-4 days to recover from the refractory condition. Cells also became refractory to GnRH when luteinizing hormone (LH) release was blocked by Ca2+ chelation. Drugs such as veratridine, ionophore A23187, or high K+, which stimulated LH release without GnRH receptor occupancy, were also capable of causing refractoriness to GnRH in long-term exposure. These data suggest that the refractory state observed after stimulation with GnRH is a result of the combined effects of a Ca2+-independent receptor-mediated mechanism for desensitization and some other postreceptor mechanism. Tunicamycin interfered with recovery, whereas cycloheximide did not. This evidence presents a potential role for protein glycosylation in the restoration of responsiveness. Phorbol myristate acetate did not cause subsequent refractoriness to GnRH, and dibutyryl adenosine 3',5'-cyclic monophosphate had no measurable effect on the rate of recovery.