Vascular endothelial growth factor (VEGF) increases vascular permeability by stimulating endothelial Ca(2+) influx. Here we provide evidence that links VEGF-mediated increased permeability and endothelial intracellular Ca(2+) concentration ([Ca(2+)](i)) with diacylglycerol (DAG)-mediated activation of the transient receptor potential channels (TRPCs). We used the Landis-Michel technique to measure changes in hydraulic conductivity (L(p)) and fluorescence photometry to quantify changes in endothelial [Ca(2+)](i) in individually perfused Rana mesenteric microvessels in vivo and transfected nonendothelial cells in vitro. The membrane-permeant DAG analog 1-oleoyl-2-acetyl-sn-glycerol (OAG, 100 microM), which is known to increase Ca(2+) influx through TRPCs, transiently increased L(p) 3.8 +/- 1.2-fold (from 1.6 +/- 0.8 to 9.8 +/- 2.7 x 10(-7) cm.s(-1).cmH(2)O(-1); P < 0.0001; n = 18). Protein kinase C inhibition by bisindolylmaleimide (1 microM) did not affect the OAG-induced increases in L(p). OAG also significantly increased microvascular endothelial [Ca(2+)](i) in vivo (n = 13; P < 0.0001), which again was not sensitive to protein kinase C inhibition. VEGF induced a transient increase in endothelial [Ca(2+)](i) in human embryonic kidney cells (HEK-293) that were cotransfected with VEGF receptor 2 and TRPC-6 but not with control, VEGF receptor 2, or TRPC-6 expression vector alone (P < 0.01; n = 9). Flufenamic acid, which has been shown to enhance activity of TRPC-6 but inhibit TRPC-3 and -7, enhanced the VEGF-mediated increase in L(p) in approximately half of the vessels tested but inhibited the response in the other half of the vessels. These data provide evidence consistent with the hypothesis that VEGF increases vascular permeability via DAG-mediated Ca(2+) entry through TRPCs. Although the exact identities of the TRPCs remain to be confirmed, TRPC-6 appears to be a likely candidate in approximately half of the vessels.