Recently, we showed that neutral sphingomyelinase (N-SMase) is concentrated at the endothelial cell surface in caveolae and is activated to produce ceramide in an acute and transient manner by increase in flow rate and pressure in rat lung vasculature (Czarny, M., Liu, J., Oh, P., and Schnitzer, J. E., 2003, J Biol Chem 278, 4424-4430). Here, we report further on our investigations of this new acute mechanotransduction pathway. We employed three different experimental models to explore the role of N-SMase and ceramides in mechanosignaling: 1) a cell-free, in vitro model using isolated luminal plasma membranes of rat lung endothelium; 2) a fluid shear stress model using monolayers of intact bovine aorta endothelial cell in culture; and 3) an in situ model using controlled perfusion of the rat lung vasculature. Scyphostatin, which specifically inhibited neutral but not acid sphingomyelinase activity, prevented mechano-activation of neutral sphingomyelinase as well as downstream tyrosine and mitogen activated protein kinases. Cell permeable ceramide analogs (N-acetylsphingosine, C2-ceramide and N-hexanoylsphingosine, C6-ceramide) but not the inactive, dihydro-derrivatives D2-ceramide and D6-ceramide (N-acetylsphinganine and N-hexanyolsphinganine, respectively) mimic rapid mechano-induced tyrosine phosphorylation of cell surface proteins, as well as mechano-activation of Src-like kinases and the extracellular regulated kinase pathway. The responses common to ceramide and mechanical stress were inhibited by genistein, herbamycin A, and PP2, but not PP3 which suggests an obligate role of Src-like kinases in ceramide-mediated mechanotransduction. Ceramides also induce serine/threonine phosphorylation to activate the Akt/endothelial nitric oxide synthase pathway. Thus, N-SMase at the plasma membrane in caveolae may be an upstream initiating mechanosensor, which acutely triggers mechanotransduction by generation of the lipid second messenger, ceramide.