Factors that determine the interaction of ouabain with the positive inotropic receptor were examined in isolated perfused guinea-pig hearts. The hearts were exposed to ouabain during a perfusion with a modified Krebs-Henseleit solution. The interaction of ouabain with the inotropic receptors under those conditions was estimated by subsequently perfusing the heart with a regular Krebs-Henseleit solution and monitoring the resting and developed tension. Ouabain failed to cause an increase in the force of contraction when the cardiac tissue was exposed to this agent in the absence of Na+ and Ca2+ either in the presence of a low or high concentration of K+. The absence of Ca2+ or the lack of contraction was not responsible for the failure of ouabain to interact with the inotropic receptor, since the exposure of cardiac tissue to ouabain in a Ca,+-free medium containing Na+ resulted in a development of the positive inotropic effect. Thus, ouabain does not interact with its inotropic receptor in the absence of Na+. The properties of the ouabain-receptor interaction resemble those of ouabain binding to Na+, K+-ATPase. In addition, ouabain increases the mobility of superficially bound Ca2+.