Caveolin-1 (Cav-1) regulates agonist-induced Ca(2+) entry in endothelial cells; however, how Cav-1 regulates this process is poorly understood. Here, we describe that Cav-1 scaffold domain (NH(2)-terminal residues 82-101; CSD) interacts with transient receptor potential canonical channel 1 (TRPC1) and inositol 1,4,5-trisphosphate receptor 3 (IP(3)R3) to regulate Ca(2+) entry. We have shown previously that the TRPC1 COOH-terminal residues 781-789 bind to CSD. In the present study, we show that the TRPC1 COOH-terminal residues 781-789 truncated (TRPC1-CDelta781-789) mutant expression abolished Ca(2+) store release-induced Ca(2+) influx in human dermal microvascular endothelial cell line (HMEC) and human embryonic kidney (HEK-293) cells. To understand the basis of loss of Ca(2+) influx, we determined TRPC1 binding to IP(3)R3. We observed that the wild-type (WT)-TRPC1 but not TRPC1-CDelta781-789 effectively interacted with IP(3)R3. Similarly, WT-TRPC1 interacted with Cav-1, whereas TRPC1-CDelta781-789 binding to Cav-1 was markedly suppressed. We also assessed the direct binding of Cav-1 with TRPC1 and observed that the WT-Cav-1 but not the Cav-1DeltaCSD effectively interacted with TRPC1. Since the interaction between TRPC1 and Cav-1DeltaCSD was reduced, we measured Ca(2+) store release-induced Ca(2+) influx in Cav-1DeltaCSD-transfected cells. Surprisingly, Cav-1DeltaCSD expression showed a gain-of-function in Ca(2+) entry in HMEC and HEK-293 cells. We observed a similar gain-of-function in Ca(2+) entry when Cav-1DeltaCSD was expressed in lung endothelial cells of Cav-1 knockout mice. Immunoprecipitation results revealed that WT-Cav-1 but not Cav-1DeltaCSD interacted with IP(3)R3. Furthermore, we observed using confocal imaging the colocalization of IP(3)R3 with WT-Cav-1 but not with Cav-1DeltaCSD on Ca(2+) store release in endothelial cells. These findings suggest that CSD interacts with TRPC1 and IP(3)R3 and thereby regulates Ca(2+) store release-induced Ca(2+) entry in endothelial cells.