We investigated the possibility that the TRPC gene family of putative store-operated Ca2+entry channels contributes to the increase in microvascular endothelial permeability by prolonging the rise in intracellular Ca2+signaling. Studies were made in wild-type (wt) and TRPC4 knockout (TRPC4−/−) mice and lung vascular endothelial cells (LECs) isolated from these animals. RT-PCR showed expression of TRPC1, TRPC3, TRPC4, and TRPC6 mRNA in wt LECs, but TRPC4 mRNA expression was not detected in TRPC4−/−LECs. We studied the response to thrombin because it is known to increase endothelial permeability by the activation of G protein-coupled proteinase-activated receptor-1 (PAR-1). In wt LECs, thrombin or PAR-1 agonist peptide (TFLLRNPNDK-NH2) resulted in a prolonged Ca2+transient secondary to influx of Ca2+. Ca2+influx activated by thrombin was blocked by La3+(1 &mgr;mol/L). In TRPC4−/−LECs, thrombin or TFLLRNPNDK-NH2produced a similar initial increase of intracellular Ca2+secondary to Ca2+store depletion, but Ca2+influx induced by these agonists was drastically reduced. The defect in Ca2+influx in TRPC4−/−endothelial cells was associated with lack of thrombin-induced actin-stress fiber formation and a reduced endothelial cell retraction response. In isolated-perfused mouse lungs, the PAR-1 agonist peptide increased microvessel filtration coefficient (Kf,c), a measure of vascular permeability, by a factor of 2.8 in wt and 1.4 in TRPC4−/−; La3+(1 &mgr;mol/L) addition to wt lung perfusate reduced the agonist effect to that observed in TRPC4−/−. These results show that TRPC4-dependent Ca2+entry in mouse LECs is a key determinant of increased microvascular permeability.