In cultured porcine aortic endothelial monolayers, the effect of ATP on myosin light chain (MLC) phosphorylation, which controls the endothelial contractile machinery, was studied. ATP (10 microM) reduced MLC phosphorylation but increased cytosolic Ca(2+) concentration ([Ca(2+)](i)). Inhibition of the ATP-evoked [Ca(2+)](i) rise by xestospongin C (10 microM), an inhibitor of the inositol trisphosphate-dependent Ca(2+) release from endoplasmic reticulum, did not affect the ATP-induced dephosphorylation of MLC. MLC dephosphorylation was prevented in the presence of calyculin A (10 nM), an inhibitor of protein phosphatases PP-1 and PP-2A. Thus ATP activates MLC dephosphorylation in a Ca(2+)-independent manner. In the presence of calyculin A, MLC phosphorylation was incremented after addition of ATP, an effect that could be abolished when cells were loaded with the Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N, N,N',N'-tetraacetic acid acetoxymethyl ester (10 microM). Thus ATP also activates a Ca(2+)-dependent kinase acting on MLC. In summary, ATP simultaneously stimulates a functional antagonism toward both phosphorylation and dephosphorylation of MLC in which the dephosphorylation prevails. In endothelial cells, ATP is the first physiological mediator identified to activate MLC dephosphorylation by a Ca(2+)-independent mechanism.