Cyclic nucleotide phosphodiesterase (PDE) activities were characterized in the cytosolic and post-nuclear membrane preparations of guinea pig cardiac ventricles. The cytosolic PDE activities were stimulated 5-fold by calmodulin (CaM) on both substrates (1 microM) and 1.2-fold by cGMP (5 microM) on cAMP hydrolysis. Conversely, in the membrane preparation, CaM only stimulated PDE activities 1.2- to 1.4-fold, but cGMP induced a 3-fold increase of the hydrolysis of cAMP. In both the cytosolic and the membrane preparations, the hydrolysis of cAMP was inhibited by 100 microM of either the PDE III inhibitor SK&F 94120 (27% and 31% respectively) or the PDE IV inhibitor rolipram (14% and 23% respectively). Four peaks were resolved from the cytosolic preparation by chromatography. Peak A and peak B hydrolyzed both cAMP and cGMP and were stimulated respectively by CaM and cGMP. Peak C and peak D selectively hydrolyzed cAMP. Peak C had an apparent Km value for cAMP of 3.3 microM and was inhibited by PDE IV inhibitors. Peak D showed an apparent Km value for cAMP of 0.43 microM and was inhibited by cGMP and by cardiotonic inhibitors of PDE III. Similar potencies of these inhibitors were observed in the membrane preparation. These results suggest that in guinea pig cardiac ventricles: (1) PDE I (CaM-activated) is almost exclusively cytosolic; (2) PDE II (cGMP-stimulated), PDE III (cGMP-inhibited and cardiotonic-sensitive) and PDE IV (rolipram-sensitive) are present in cytosolic and membrane preparations; (3) PDE III and PDE IV differ in their apparent Km values for cAMP. The latter observation could explain the differential effects of PDE III and PDE IV inhibitors in the regulation of cardiac contraction.