Sustained exposure of gonadotropes to GnRH causes a pronounced desensitization of gonadotropin release, but the mechanisms involved are poorly understood. It is known that desensitization is associated with decreased GnRH-R and Gq/11 levels in T3-1 cells, but it is not known whether downstream signaling is impaired. We have shown previously that chronic stimulation of signaling via expression of an active form of Gq causes GnRH resistance in LT2 cells. In this study we investigated whether chronic GnRH treatment could down-regulate PKC, cAMP, or Ca²⁺-dependent signaling in LT2 cells. We found that chronic GnRH treatment desensitizes cells to acute GnRH stimulation not only by reducing GnRH receptor and Gq/11 expression, but also by downregulating PKC, cAMP, and calcium-dependent signaling. Desensitization was observed for activation of ERK and p38 MAPK, and induction of c-fos and LH protein expression. Activation of individual signaling pathways was able to partially mimic the desensitizing effect of GnRH on ERK, p38 MAPK, c-fos and LH, but not on Gq/11. Chronic stimulation with phorbol esters reduced GnRH receptor expression to the same extent as chronic GnRH. Sustained GnRH also desensitized PKC signaling by downregulating the , , and isoforms of PKC. We further show that chronic GnRH treatment causes heterologous desensitization of other Gq-coupled receptors.