The mechanism by which mechanical stress induces nitric oxide (NO) synthesis in endothelium is still controversial. Hypotonic stress (HTS, -20%) induced ATP release, which evoked Ca2+ transients in bovine aortic endothelial cells (BAEC), HTS also induced NO synthesis, assessed by DAF-S fluorescence, which was suppressed by inhibiting endogenous ATP-induced Ca2+ transients with suramin or neomycin, Exogenously applied ATP mimicked these responses. Pretreatment with wortmannin did not affect DAF-2 fluorescence, suggesting that Akt phosphorylation was not involved in MTS-induced NO synthesis, These results indicate that endogenous ATP plays a central role in HTS-induced NO synthesis in BAEC.