To elucidate the positive inotropic mechanism of levosimendan, we set out to perform a sequence of tests in which we characterized the concentration dependences of the putative subcellular effects of levosimendan. The Ca2+-sensitizing potentials of levosimendan were determined in demembranated guinea pig myocytes at a steady [Ca2+] in order to avoid interference with the PDE isoenzymes and ionic channels. Since simultaneous inhibition of two PDE isoenzymes (i.e. PDE III and PDE IV) has been shown to be a prerequisite of a biologically effective elevation in intracellular [cAMP] in the myocardium, the IC50 values for the inhibitory activities of levosimendan on the isolated PDE III and PDE IV isoforms were also determined. Additionally, we assessed the potencies and efficacies of the two molecules on the left ventricular performance in Langendorff-perfused isolated guinea pig hearts at constant heart rate and coronary flow. We found well-pronounced similarities in the concentration dependences of the Ca2+-sensitizing and positive inotropic actions of levosimendan. The inhibition of PDE IV required significantly higher concentrations of levosimendan than those measured in the human plasma (6 nM or 12 nM, respectively) following the therapeutic administration of the compound. This finding may explain why the myoplasmic [cAMP] might remain unaltered during the development of levosimendan-induced positive inotropy. Our data support the hypothesis that levosimendan exerts its positive inotropy via a Ca2+-sensitizing mechanism and not via simultaneous inhibition of the PDE III and PDE IV isozymes in the myocardium at their maximal free plasma concentrations.