Isoenzyme-specific phosphodiesterase (PDE) inhibitors are potential positive inotropic drugs. For evaluating such drugs in experimental models and to understand the physiological roles of the different isoenzymes, it is necessary to know what isoenzymes are present in the tissues studied. Rat myocardium has been reported to be devoid of the particulate cGMP-inhibited cAMP-PDE (type III isoenzyme). Here we re-evaluate the isoenzyme profile of rat myocardium. The cAMP-PDE isoenzyme patterns were studied by ion-exchange chromatography using siguazodan and rolipram, specific inhibitors of type III and IV isoenzymes, respectively. In contrast to earlier reports, type III isoenzyme was abundant in the particulate fraction. PDE III-specific antibodies depressed PDE activity and stained bands in Western blot with molecular masses 64 and 71 kDa. Type III isoenzyme of myocardial membranes was found to be unstable at 37 degrees C which may explain why earlier investigators have failed to demonstrate its presence. The data presented in this paper show that rat heart particulate fraction contains two low Km PDE isoenzymes, type III and type IV, in equal activities. Thus, in contrast to previous reports, this paper clearly shows the presence of considerable amounts of membrane-bound type III PDE isoenzyme in rat myocardium.