Regulators of G protein signaling (RGS) play a pivotal role in cellular signal transduction. RGS3 or RGS10 were overexpressed in GGH(3) cells [GH(3) cells stably expressing the GnRH receptor (GnRHR)]. Responsiveness to a GnRH agonist was assessed because RGS proteins attenuate production of inositol phosphates (IP) and/or cAMP, molecules believed to be involved in GnRH signaling. In addition, site-directed mutagenesis of a potentially palmitoylated Cys(60) residue of RGS10 was used to assess the significance of this site. We observed maximum inhibition of GnRH-stimulated IP responses by RGS3 and by the conserved domain of RGS10 at both 48 and 72 h after transfection, indicating their involvement in G(q)alpha mediated signaling. Significantly diminished cAMP production was observed at all times when cells overexpressed the conserved domain of RGS10; no effect was observed with RGS3 on G(s)alpha-mediated signaling. Palmitic acid incorporation into RGS3 was dependent on agonist occupancy of GnRHR, whereas palmitoylation of RGS10 was constitutive. Mutation of the conserved Cys(60) residue of RGS10 obviated its negative regulatory action on GnRH-stimulated responses, indicating that this site is crucial for its activity on this system. This study is the first demonstration of a role for palmitoylation of this conserved Cys(60) in mammalian G protein signaling.