Several mechanisms have been proposed to explain the acute cardiovascular effects elicited by androgens, such as vasodilation and positive inotropism. Phosphodiesterases (PDEs) are important modulators of cardiac contractility. However, an effect on PDEs by androgens in cardiac tissues has not previously been reported. In this study, extracts from rat ventricles and isolated left atria were assayed for cAMP-dependent PDE activity. To study the tissue selectivity, the enzymatic activity was also assayed in extracts from bovine tracheal smooth muscle and Chinese hamster ovary (CHO) cells. Functional assays were also performed with isolated atria. Testosterone, but not 5α- and 5β-dihydrotestosterone, inhibited cAMP-PDE activity in extracts from left ventricles and atria. In atria, the inhibition of cAMP-PDE activity was associated with an increase in intracellular levels of cAMP and a cardiotonic response. This effect was not elicited in tracheal muscle strips or CHO cell extracts, suggesting the possibility of tissue and cAMP-PDE selectivity. The results of these studies suggest a new mechanism of action of testosterone in the rat heart, which might contribute to the reported cardiotonic effect.