[3H]LY186126, an analogue of the cardiotonic agent indolidan, was shown to bind reversibly and with high affinity (Kd=4 nM) to a single class of binding sites within canine myocardial vesicles. Binding site density measured in various cardiac membrane fractions correlated well with Ca2+-ATPase activity (r=0.94; p<0.01), but not with Na+,K+-ATPase or azide sensitive ATPase, indicating a localization of these sites within sarcoplasmic retlculum membranes. Divalent cations were required for binding and displayed the following order of activation: Zn2++ Mn2+Mg2+Ca2+. Differential activation of [3H]LY186126 binding by various divalent cations was due to alterations in binding site density, rather than affinity. cGMP and selective inhibitors of type IV membrane-bound phosphodiesterase (SR-PDE), for example, indolidan, milrinone, imazodan, and enoxlmone, selectively displaced bound [3H]LY186126; caffeine, theophylline, and rolipram were relatively impotent as inhibitors of radiolabel binding. Kdvalues from displacement corves were highly correlated with IC50values for inhibition of SR-PDE (r=0.92; p<0.001). In addition, Kdvalues correlated well with published ED50values for increases in cardiac contractility in pentobarbital-anesthetized dogs (r=0.94; p<0.001). The results support the hypothesis that [3H]LY186126 labels the pharmacological receptor for the class of positive inotropic agents characterized as isozyme-selective phosphodiesterase inhibitors. Furthermore, the data suggest that the identity of the site labeled by [3H]LY186126 is SR-PDE, the type IV isozyme of cardiac phosphodiesterase located in the sarcoplasmic retlculum.