We characterized the effects of vanadate, an inhibitor of tyrosine phosphatase, on the tension, the level of myosin light chain (MLC) phosphorylation, and Rho A activation in intact ileal longitudinal smooth muscle of the guinea pig to study the role of tyrosine phosphorylation in contraction signaling. Vanadate exerted a sustained contraction with a slow onset of tension development, in a concentration-dependent manner. The contractile effects of vanadate were accompanied by increases in the level of MLC phosphorylation. The tyrosine kinase inhibitor genistein; the MLC kinase inhibitor 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML-9); and the Rho kinase inhibitor (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride, monohydrate (Y-27632) inhibited the vanadate-induced contraction and MLC phosphorylation. Vanadate caused Rho A translocation from the cytosol to the membrane fraction, which was inhibited by genistein, but not by ML-9 and Y-27632. These data indicate that vanadate induces Rho A activation probably via protein tyrosine phosphorylation and the subsequent contraction through increases in the level of MLC phosphorylation.