Phosphodiesterase isozymes were isolated by diethylaminoethyl ether (DEAE) column chromatography from cardiac muscle (canine, guinea pig), vascular (canine and guinea pig aortic) and airway (canine tracheal) smooth muscle. All peak I phosphodiesterases had a low apparent Km (0.29-0.49 microM) for guanosine 3':5' cyclic monophosphate (cGMP) and all peak III phosphodiesterases had a low apparent Km (0.35-0.58 microM) for adenosine 3':5' cyclic monophosphate (cAMP); trachealis peak III also had a high Km for cAMP (32 microM). The potency and selectivity for inhibition of peak I or peak III phosphodiesterase by theophylline and papaverine, the peak I selective inhibitor M + B 22948, and the peak III selective inhibitors amrinone, milrinone, imazodan, CI-930 and piroximone were approximately equal when isozymes isolated from aortic smooth muscle were compared to isozymes isolated from cardiac muscle of both species. Rolipram was relatively potent as a peak III phosphodiesterase inhibitor in canine cardiac muscle, but was impotent in the other cardiovascular peak IIIs. In tracheal smooth muscle, the cardiovascular selective peak III phosphodiesterase selective inhibitors were substantially less potent while rolipram was more potent as a peak III inhibitor. In summary, these studies show that while cardiac and vascular smooth muscle phosphodiesterase isozymes are pharmacologically similar, there is pharmacological and substrate heterogeneity of peak III phosphodiesterase in aortic vs. trachea smooth muscle within the same species.