Hydrostatic stress causes Ca2+ influx into endothelial cells, which is considered critical for loss of barrier integrity and hydrostatic edema formation. Here, we tested the role of the mechanosensitive channels TRPV2 and TRPV4 in this scenario. In the isolated perfused rat lung, we monitored endothelial Ca2+ concentration ([Ca2+ ]i) by in situ fluorescence imaging and measured the capillary filtration coefficient (Kfc ) by microgravimetry. Data are mean±SEM, *p<0.05 vs. control.Western blot analyses revealed expression of both TRPV2 and TRPV4 in freshly isolated lung microvascular endothelial cells. Activation of TRPV2 or TRPV4 by either 2-APB (200 mM) or 4aPDD (10 mM) increased lung endothelial [Ca2+ ]i within 30 min from 102±1 to 171±23* and 161±45* nM, respectively. Activation of TRPV2 or TRPV4 also increased lung Kfc from 0.4±0.1 at baseline to 1.2±0.3* and 1.1±0.2* ml min-1 cmH2O-1 100g-1 , and increased lung wet/dry weight ratio (W/D) from 5.09±0.03 to 5.96±0.05* and 6.02±0.03*, respectively. At elevated hydrostatic stress (left atrial pressure of 15 cmH2O), the inhibitor of TRPV channels, ruthenium red, prevented the increases in [Ca2+ ]i (104±3* vs. 194±7 nM) and of Kfc (0.8±0.1* vs. 2.6±0.7 ml min-1 cmH2O-1 100g-1 ), and reduced lung W/D from 6.09±0.07 to 5.80±0.11*. These findings implicate endothelial TRPV channels involved in the pathogenesis of lung hydrostatic edema.