We studied the effects of protein kinase C (PKC) activation on endothelial cell surface expression and function of the proteolytically activated thrombin receptor 1 (PAR-1). Cell surface PAR-1 expression was assessed by immunofluorescence (using anti-PAR-1 monoclonal antibody), and receptor activation was assessed by measuring increases in cytosolic Ca²⁺ concentration in human dermal microvascular endothelial cells (HMEC) exposed to -thrombin or phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). Immunofluorescence showed that thrombin and TPA reduced the cell surface expression of PAR-1. Prior exposure of HMEC to thrombin for 5 min desensitized the cells to thrombin, indicating homologous PAR-1 desensitization. In contrast, prior activation of PKC with TPA produced desensitization to thrombin and histamine, indicating heterologous PAR-1 desensitization. Treatment of cells with staurosporine, a PKC inhibitor, fully prevented heterologous desensitization, whereas thrombin-induced homologous desensitization persisted. Depletion of PKC isozymes (PKC_I and PKC_II) by transducing cells with antisense cDNA of PKC_I prevented the TPA-induced decrease in cell surface PAR-1 expression and restored ~60% of the cytosolic Ca²⁺ signal in response to thrombin. In contrast, depletion of PKC isozymes did not affect the loss of cell surface PAR-1 and induction of homologous PAR-1 desensitization by thrombin. Therefore, homologous PAR-1 desensitization by thrombin occurs independently of PKC isozymes, whereas the PKC-activated pathway is important in signaling heterologous PAR-1 desensitization in endothelial cells.