Canine and guinea-pig left ventricular muscle contains multiple molecular forms of phosphodiesterase (PDE) which vary according to substrate specificity, stimulation by calmodulin and response to various selective and nonselective phosphodiesterase inhibitors. Both species possess a cyclic AMP-specific form of phosphodiesterase (PDE III). In the dog, both soluble and particulate forms of PDE III are present. The particulate form of PDE III is potently inhibited by cyclic GMP and the selective PDE III inhibitors imazodan (CI-914) and cilostamide, but is only weakly inhibited by the selective PDE III inhibitors Ro 20-1724 and rolipram. In contrast, the soluble form of PDE III in canine left ventricle is only weakly inhibited by cyclic GMP, imazodan and cilostamide, but is potently inhibited by Ro 20-1724 and rolipram. Guinea-pig left ventricle contains only one subclass of PDE III, which is potently inhibited by cyclic GMP, imazodan and cilostamide, but not by Ro 20-1724 or rolipram. However, whereas the imazodan-sensitive subclass of PDE III is a particulate enzyme in the canine left ventricle, in the guinea-pig this subclass of PDE III is a soluble enzyme. Both soluble and particulate PDE III's are (i) insensitive to calmodulin; (ii) possess comparable Km and Vmax values for hydrolysis of cyclic AMP; (iii) are equally inhibited by the nonselective PDE inhibitor theophylline, and (iv) are eluted from DEAE-cellulose anion-exchange resin by comparable concentrations of sodium acetate. The demonstration of distinct subclasses of the cyclic AMP-specific phosphodiesterase (PDE III) in canine left ventricular muscle associated with different domains of the cell suggests compartmentation of cyclic AMP. In addition, the observation that the imazodan-sensitive form of PDE III is a particulate enzyme in canine left ventricle and a soluble enzyme in guinea-pig left ventricle may explain the species differences which exist regarding the positive inotropic response to imazodan in these two species.