An understanding of the mechanisms that regulate signaling by the substance P (SP) or neurokinin 1 receptor (NK1-R) is of interest because of their role in inflammation and pain. By using activators and inhibitors of protein kinase C (PKC) and NK1-R mutations of potential PKC phosphorylation sites, we determined the role of PKC in desensitization of responses to SP. Activation of PKC abolished SP-induced Ca²⁺ mobilization in cells that express wild-type NK1-R. This did not occur in cells expressing a COOH-terminally truncated NK1-R (NK1-R324), which may correspond to a naturally occurring variant, or a point mutant lacking eight potential PKC phosphorylation sites within the COOH tail (NK1-R Ser-338, Thr-339, Ser-352, Ser-387, Ser-388, Ser-390, Ser-392, Ser-394/Ala, NK1-RKC4). Compared with wild-type NK1-R, the t_1/2 of SP-induced Ca²⁺ mobilization was seven- and twofold greater in cells expressing NK1-R324 and NK1-RKC4, respectively. In cells expressing wild-type NK1-R, inhibition of PKC caused a 35% increase in the t_1/2 of SP-induced Ca²⁺ mobilization. Neither inhibition of PKC nor receptor mutation affected desensitization of Ca²⁺ mobilization to repeated challenge with SP or SP-induced endocytosis of the NK1-R. Thus PKC regulates SP-induced Ca²⁺ mobilization by full-length NK1-R and does not regulate a naturally occurring truncated variant. PKC does not mediate desensitization to repeated stimulation or endocytosis of the NK1-R.