1. Cantharidin, an inhibitor of protein phosphatase types 1 (PP1) and 2A (PP2A), increased basal tone of bovine isolated coronary artery rings (CARs) with and without endothelium in a time- and concentration-dependent manner with pEC50 values of about 5.1 and 5.2, respectively, for both preparations. 2. Beta-Adrenoceptor stimulation with isoprenaline (Iso; 0.03-100 microM) or inhibition of phosphodiesterase activity by 3-isobutyl-1-methylxanthine (IBMX; 10-1000 microM), respectively, relaxed CARs precontracted with KCl (75 mM). CARs with and without endothelium showed no difference in the relaxing response to Iso and IBMX, respectively. 3. Cantharidin (3 microM) attenuated vasorelaxation induced by Iso (0.03-100 microM) in CARs with and without endothelium in a time-dependent manner, whereas vasorelaxation induced by IBMX (10-1000 microM) was not attenuated by 3 microM cantharidin. 4. Cantharidin (3 microM) did not affect cyclic AMP content in bovine cultured vascular cells, i.e. coronary artery smooth muscle cells (BCs), aortic endothelial cells (BAECs) and aortic smooth muscle cells (BASMCs), either under basal conditions, after beta-adrenoceptor stimulation (Iso) or inhibition of phosphodiesterase activity (IBMX), respectively. 5. Cantharidin inhibited protein phosphatase activity in homogenates from bovine coronary artery rings with a pIC50 of about 6.0. In homogenates of bovine cultured vascular cells pIC50 values of cantharidin amounted to about 6.5 for BCs, 6.7 for BAECs and 6.7 for BASMCs, respectively. 6. It was concluded that cantharidin differently affects vasorelaxation due to stimulation of beta-adrenoceptors (Iso) or inhibition of phosphodiesterase activity (IBMX), respectively. The attenuation of beta-adrenoceptor-mediated vasorelaxation by phosphatase inhibition is not due to diminished adenosine 3':5'-cyclic monophosphate (cyclic AMP) generation but could be evidence for different subcellular compartments of cyclic AMP.