This study reports the isolation and characterization of cyclic nucleotide phosphodiesterases (PDEs) associated with membrane fraction in comparison to cytosolic forms from bovine aorta. DEAE-Sephacel chromatography of a solubilized membrane fraction from a homogenate, prepared under isotonic conditions in the presence of protease inhibitors, yielded one major peak of PDE activity that specifically hydrolyzed cAMP and was not stimulated by calmodulin: It appeared to contain two subtypes of PDE. The first subtype belonged to the cyclic GMP (cGMP)-inhibited PDE family, (PDE III): It had an apparent Km value of 0.4 microM and was potently inhibited by cGMP, LY186126, and cilostamide. The second was a rolipram-sensitive PDE form (PDE IV) that had an apparent Km value for cAMP hydrolysis of 1.1 microM, was selectively inhibited by rolipram and denbufylline, and was insensitive to cGMP. These two forms had kinetic and pharmacologic profiles similar to those resolved by DEAE-Sephacel from the cytosolic fraction (105,000 g supernatant). In addition, DEAE-Sephacel chromatography of the cytosolic fraction revealed another peak of PDE activity that could be resolved with high-performance liquid chromatography (HPLC) into a calmodulin-sensitive form that preferentially hydrolyzed cGMP (PDE I) and a calmodulin-insensitive form that specifically hydrolyzed cGMP (PDE V). The presence of a PDE III in vascular smooth muscle that exhibited similarities to the cGMP-inhibited PDE from cardiac tissues, the target of several new cardiotonic agents, suggests that a single mechanism of action may account for the cardiotonic and vasodilating properties of PDE III inhibitors.