Cantharidin concentration dependently increased the force of contraction in isolated guinea pig papillary muscles (1-100 microM). The positive inotropic effect is accompanied by a reduction in time to peak tension and relaxation time. Cantharidin did not exert a positive chronotropic effect in spontaneously beating right atria. L-type calcium channel currents of guinea pig cardiomyocytes were moderately increased by cantharidin (by about 20%), both at the whole-cell level (2 mM Ca2+) and at the single channel level (70 mM Ba2+). There was a correspondingly small increment of single channel availability. Additionally, a larger proportion of single-channel sweeps displayed high open probability-gating (so-called mode 2-gating). Cantharidin inhibited both type 1 and type 2A phosphatase activity in phosphatases purified from guinea pig ventricles [IC50 2.70 (2.06-3.53) and 0.13 (0.05-0.34) microM, n = 5-6, with 95% confidence intervals, respectively]. In isolated [32P]-labeled guinea pig ventricular cardiomyocytes, cantharidin (10 microM) increased the phosphorylation state of phospholamban (to 210% of control), the inhibitory subunit of troponin (to 155% of control), C-protein (to 156% control) and various additional proteins. It is concluded that the effects of cantharidin are likely mediated by increasing the phosphorylation state of several regulatory proteins. Furthermore, cantharidin might be an economical tool to investigate the function of phosphatases in model organ systems.