In this study, site-directed mutagenesis of potential phosphorylation sites (Thr238, Ser253, and Thr264) for protein kinase C and C-terminal portion (Ala260-Leu265) of the third intracellular loop of the rat GnRH receptor (rGnRHR) was performed to assess the significance of these regions in the function of the GnRHR. Mutation at one or all of the three potential phosphorylation sites had differential effects on receptor ligand binding. Mutation of Ser253 or Thr264 to Ala did not significantly affect the receptor-binding affinity but decreased the number of measurable binding sites. Mutation of Thr238 to Ala or triple mutation of Thr238, Ser253, and Thr264 impaired or abolished receptor-binding affinity. Mutations of the potential phosphorylation sites affected receptor-mediated inositol phospholipid (IP) production and correlated with alterations in receptor binding after mutation, but they did not significantly affect receptor-mediated cAMP production or cAMP-mediated prolactin release. In addition, mutation of Ser253 or Thr264 to Ala did not affect the GnRH-provoked desensitization in terms of GnRH agonist-stimulated IP production. Deletion of the C-terminal portion (Ala260-Leu265) of the third intracellular loop of the rGnRHR, including a potential phosphorylation site (Thr264), abolished the receptor-binding affinity and receptor-mediated signal transduction. Replacement of the deleted C-terminal portion with a C-terminal portion (Ala-Ala-Arg-Thr-Leu-Ser) of the third intracellular loop of the Gq/11-coupled rat M1 muscarinic acetylcholine receptor did not restore receptor function. These results suggest that the potential phosphorylation sites or the region around the phosphorylation site of the third intracellular loop of the GnRHR is important for the structural integrity and expression of the receptor but that phosphorylation at these sites is not required for desensitization.