Phosphorylation-deficient serotonin 5-HT(2C) receptors were generated to determine whether phosphorylation promotes desensitization of receptor responses. Phosphorylation of mutant 5-HT(2C) receptors that lack the carboxyl-terminal PDZ recognition motif (Ser(458)-Ser-Val-COOH; DeltaPDZ) was not detectable based on a band-shift phosphorylation assay and incorporation of (32)P. Treatment of cells stably expressing DeltaPDZ or wild-type 5-HT(2C) receptors with serotonin produced identical maximal responses and EC(50) values for eliciting [(3)H]inositol phosphate formation. In calcium imaging studies, treatment of cells expressing DeltaPDZ or wild-type 5-HT(2C) receptors with 100 nm serotonin elicited initial maximal responses and decay rates that were indistinguishable. However, a second application of serotonin 2.5 min after washout caused maximal responses that were approximately 5-fold lower with DeltaPDZ receptors relative to wild-type 5-HT(2C) receptors. After 10 min, responses of DeltaPDZ receptors recovered to wild-type 5-HT(2C) receptor levels. Receptors with single mutations at Ser(458) (S458A) or Ser(459) (S459A) decreased serotonin-mediated phosphorylation to 50% of wild-type receptor levels. Furthermore, subsequent calcium responses of S459A receptors were diminished relative to S458A and wild-type receptors. These results establish that desensitization occurs in the absence of 5-HT(2C) receptor phosphorylation and suggest that receptor phosphorylation at Ser(459) enhances resensitization of 5-HT(2C) receptor responses.