Okadaic acid exerts a positive inotropic effect in cardiac preparations. We studied whether the positive inotropic effect of okadaic acid in cardiac preparations could be due to phosphatase inhibition and whether this inhibition affects the phosphorylation of cardiac proteins. In papillary muscles from guinea pigs, 30 microM okadaic acid increased force of contraction to 175% of predrug value. In isolated guinea pig ventricular cardiomyocytes, okadaic acid augmented single Ca(2+)-channel currents by enhancing channel availability. In homogenates from ventricles, 1 microM okadaic acid completely inhibited phosphorylase a phosphatase activity. In isolated 32P-labeled ventricular cardiomyocytes, 30 microM okadaic acid increased phosphorylation of phospholamban (PLB) and troponin inhibitor (TnI) to 325 and 284% of control, respectively. Furthermore, 30 microM okadaic acid increased phosphorylation of a hitherto unknown 23-kDa protein to 352% of control. It is concluded that the effects of okadaic acid could be mediated by increasing the phosphorylation state of several proteins including PLB, a 23-kDa protein, and TnI.