1. The aim of this study was to investigate the mechanism(s) of the vasoconstrictor effect of cantharidin in bovine preparations. 2. Catalytic subunits of protein phosphatase type 1 (PP 1) and type 2A (PP 2A) were immunologically identified in coronary arteries, isolated smooth muscle cells and ventricular myocardium. 3. The mRNAs coding for catalytic subunits of PP 1alpha, PP 1beta and PP 2Aalpha were identified by hybridization with specific cDNA-probes in total RNA from coronary arteries, isolated smooth muscle cells and ventricles. 4. The activities of catalytic subunits of PP 1 and PP 2A separated by column chromatography from coronary arteries, isolated smooth muscle cells and ventricles were inhibited by cantharidin in a concentration-dependent manner. 5. Cantharidin increased the phosphorylation state of smooth muscle proteins including the regulatory light chains of myosin in 32P-labelled intact smooth muscle cells in a concentration-dependent manner. 6. Cantharidin did not affect cytosolic calcium concentrations in aortic smooth muscle cells. 7. It is suggested that cantharidin contracts smooth muscle preparations by increasing the phosphorylation state of regulatory proteins due to inhibition of phosphatase activities. Thus, cantharidin might be a useful tool to study the function of phosphatases in smooth muscle.