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 μmol/L). In TRPC4^−/− LECs, thrombin or TFLLRNPNDK-NH₂ produced 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 (K_f,c), a measure of vascular permeability, by a factor of 2.8 in wt and 1.4 in TRPC4^−/−; La³⁺ (1 μmol/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.