Desensitization of the pituitary gonadotrope was examined using perifusion of immobilized dispersed cells. Specifically, a relatively brief (20 min) pulse of GnRH was shown to result in markedly reduced release of LH in response to subsequent administration of the releasing hormone. Derivatives of cAMP exerted neither an inhibitory nor a stimulatory effect on desensitization. Nonreceptor mediated secretagogues (phorbol dibutyrate and melittin) promoted gonadotropin release without desensitizing pituitary cells indicating that release and desensitization are dissociable. However, conditions that promote GnRH receptor-receptor cross-linking did lead to reduced GnRH-responsiveness, even when the receptor was occupied by an antagonist. Cells were perifused with a conjugate consisting of two dimers of a pure GnRH-antagonist (D-pGlu1-D-Phe2-D-Trp3-D-Lys6-GnRH) attached to cross-reacting antibody. The antagonist conjugate, now able to cross-link (microaggregate) GnRH receptors, is converted to an agonist. In the present work we show that the conjugate stimulates desensitization in addition to release of LH. This observation suggests that microaggregation is the final step common to both pathways, on the one hand leading to Ca2+ mobilization and release of LH, and on the other, leading to Ca2+ independent receptor-mediated desensitization.