Platelet-activating factor (PAF) increases lung vascular permeability within minutes by activation of acid sphingomyelinase (ASM) and a subsequent nitric oxide (NO)-inhibitable and Ca 2+-dependent loss in barrier function. Objectives: To elucidate the molecular mechanisms underlying this response. Methods: In isolated perfused rat and mouse lungs, endothelial Ca 2+concentration ([Ca 2+] i) was quantified by real-time fluorescence imaging, and caveolae of endothelial cells were isolated and probed for Ca 2+ entry channels. Regulation of transient receptor potential classical (TRPC) 6-mediated currents in lung endothelial cells was assessed by patch clamp technique. Measurements and Main Results: PAF increased lung weight gain and endothelial [Ca 2+] i. This response was abrogated by inhibitors of ASM or in ASM-deficient mice, and replicated by lung perfusion with exogenous ASM or C2-ceramide. PAF increased the caveolar abundance of TRPC6 channels, which was similarly blocked by ASM inhibition. PAF-induced increases in lung endothelial [Ca 2+] i, vascular filtration coefficient, and edema formation were attenuated by the TRPC inhibitor SKF96365 and in TRPC6-deficient mice, whereas direct activation of TRPC6 replicated the [Ca 2+] i and edema response to PAF. The exogenous NO donor PapaNONOate or the cyclic guanosine 3′,5′- monophosphate analog 8Br-cGMP blocked the endothelial [Ca 2+] i and permeability response to PAF, in that they directly blocked TRPC6 channels without interfering with their PAF-induced recruitment to caveolae. Conclusions: The present findings outline a new signaling cascade in the induction of PAF-induced lung edema, in that stimulation of ASM causes recruitment of TRPC6 channels to caveolae, thus allowing for Ca 2+ influx and subsequent increases in endothelial permeability that are amplified in the absence of endothelial NO synthesis. Copyright © 2012 by the American Thoracic Society.