Classically, acute uncoupling of G protein-linked receptors has been presented as a nonselective process in which modification of the receptor by phosphorylation leads to reduction in or the loss of coupling to all effectors. Investigation of multiple signaling pathways has modified this view: for example, when expressed in Ltk- fibroblasts, the 5-hydroxytryptamine 1A (5-HT1A) receptor couples to both stimulation of intracellular calcium mobilization (via inositol phosphate generation) and inhibition of cAMP accumulation. Acute pretreatment for 2 min with 100 nM 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C (PKC), abolished the intracellular calcium response induced by 100 nM 5-HT but did not reduce 5-HT1A receptor-mediated inhibition of cAMP. In the present report, mutant 5-HT1A receptors lacking one, two, or three putative PKC phosphorylation sites located in the receptor's third cytoplasmic loop were constructed and transfected separately into Ltk- cells. The receptor mutants displayed agonist affinities similar to that of the nonmutated receptor. The relative magnitudes of 5-HT-induced changes in intracellular calcium or forskolin-stimulated cAMP levels were also similar to those of the wild-type receptor for all except one of the mutants. In addition, TPA treatment did not change the magnitude or potency of 5-HT-induced inhibition of prostaglandin E1-stimulated cAMP accumulation in the wild-type or triple-mutant 5-HT1A receptor clones. On pretreatment with TPA, there was a progressive recovery to 74% of the control 5-HT-induced increase in calcium mobilization as PKC sites were eliminated from the receptor, indicating that multiple phosphorylation sites are required for PKC-mediated receptor uncoupling. Also, these results indicate that receptor phosphorylation selectively uncouples the 5-HT1A receptor from enhancement of calcium mobilization without reducing receptor-mediated inhibition of cAMP. Thus, phosphorylation by PKC can sculpt receptor signaling via pathway-selective uncoupling.