Cysteinyl leukotrienes activate the cysteinyl leukotriene type 1 receptor (CysLT1R) to regulate numerous cell functions important in inflammatory processes and diseases such as asthma. Despite its physiologic importance no studies to date have examined the regulation of CysLT1R signaling or trafficking. We have established model systems for analyzing recombinant human CysLT1R and find regulation of internalization and signaling of the CysLT1R to be unique among GPCRs. Rapid and profound LTD4-stimulated internalization was observed for the wild type (wt) CysLT1R, whereas a C-terminal truncation mutant exhibited impaired internalization yet signaled robustly, and suggested a region within amino acids 309-321 as critical to internalization. Co-expression of arrestin2 or arrestin3 increased agonist-stimulated internalization of wt CysLT1R while inhibiting phosphoinositide (PI) production. However, co-expression of dominant negative arrestins minimally affected internalization, and wt CysLT1R internalized in murine embryonic fibroblasts lacking both arrestin2 and arrestin3, suggesting that arrestins are not the primary physiologic regulators of CysLT1Rs. Instead, pharmacological inhibition of PKC profoundly inhibited CysLT1R internalization while greatly increasing PI production by LTD4, yet had almost no effect on H1 histamine receptor internalization or signaling. Moreover, mutation of putative PKC phosphorylation sites within the CysLT1R C-tail (CysLT1RS(313-316)A) reduced receptor internalization and increased PI production by LTD4, and significantly attenuated the effects of PKC inhibition. Heterologous desensitization by PKC was also observed, as pretreatment with phorbol ester caused a small but significant reduction in PI production with subsequent LTD4 challenge in cells expressing wt CysLT1R but not CysLT1RS(313-316)A. These findings characterize the CysLT1R as the first GPCR identified to date in which PKC is the principal regulator of rapid agonist-dependent internalization and desensitization.