Vanadate inhibited K+-activated and K+-activated ouabain-sensitive p-nitrophenyl phosphatases of rat myometrium at nanomolar concentrations. The vanadate concentrations required for 50% inhibition were 220 ± 30 nM for the K+-activated component of this enzyme and 200 ± 30 nM for the K+-activated ouabain-sensitive component. Micromolar concentrations of vanadate inhibited acid and alkaline p-nitrophenyl phosphatases. ATP-dependent Ca uptake by the plasma membrane vesicles was not inhibited by 10 nM – 1 mM vanadate. Mg2+-ATPase was also not affected. Thus K+-activated and K+-activated ouabain-sensitive p-nitrophenyl phosphatase activities of the plasma membrane were most sensitive to inhibition by vanadate. Preliminary experiments demonstrated that similar to ouabain, vanadate inhibited potassium-induced abolition of spontaneous contractile activity of isolated rat myometrium in K-free Krebs. This effect of vanadate is consistent with vanadate inhibition of K+-activated ouabain-sensitive p-nitrophenyl phosphatase.