The cellular and molecular mechanisms of gonadotrope desensitization are unknown but transduction of the GnRH signal is known to involve sequentially the GnRH receptor, Gq alpha protein, phospholipase C beta-1, inositol-1,4,5-trisphosphate (IP3), and intracellular Ca+2 release. Here, we report the results of studies of a new family of proteins known as regulators of G protein signaling (RGS) that recently have been implicated in desensitization of several ligand induced processes. Using DNA-mediated transfection, we co-expressed the GnRH receptor and RGS1,2,3, or 4 in COS-1 cells. Control cells and those expressing RGS1,2, and 4 produced five fold increases in IP3 levels during the 30 sec after treatment with GnRH. In contrast, RGS3 expression suppressed by 75% the GnRH-induced IP3 responses. RGS3 was shown to bind Gq alpha protein in a model in vitro system: recombinant RGS3-glutathione-S-transferase (GST) fusion protein bound five-fold more 35S-met labeled Gq alpha protein than did with GST alone, suggesting that the mechanism of RGS3 action is attenuation of Gq alpha protein activation of phospholipase C. RGS3 mRNA and protein were observed to be expressed endogenously in the gonadotropic alpha T3-1 cell line. These results suggest a potential role for RGS3 in modulating the LH secretory responsiveness of the pituitary gonadotrope to GnRH.