The inotropic dose-response curve of ouabain in rat cardiac ventricular strips exceeded a concentration range of two decades (1 X 10(-7) M to 3 X 10(-5) M) displaying an intermediate plateau phase. In guinea-pig ventricular strips the inotropic ouabain concentrations spanned only one decade (1 X 10(-7) M-1 X 10(-6) M). Ouabain-intoxication in guinea-pig ventricular strips occurring at 3 X 10(-6) M consisted of arrhythmia and contracture, while in rat ventricular strips at the toxic concentration of 1 X 10(-4) M only a progressive increase in diastolic tension was observed. By means of atomic absorption spectroscopy the ouabain-induced loss of cellular potassium and gain of sodium in rat ventricular strips was detected only at concentrations of ouabain higher than 10(-4) M. Ouabain reduced the activity of Na/K-ATPase prepared from rat and guinea-pig cardiac ventricles to half of its maximum at 6.5 X 10(-5) M in rat and 1.0 X 10(-6) M in guinea-pig, rat heart Na/K-ATPase thus being about 60 fold less sensitive towards ouabain. Specific [3H]-ouabain binding to membrane suspensions prepared from rat and guinea-pig ventricles was characterized by a similar affinity in rat (KD = 4 X 10(-8) M) and guinea-pig (KD = 13 X 10(-8) M). The number of ouabain binding sites in rat membranes was only about 10% of the number found in guinea-pig membranes. In rat the presence of additional ouabain-binding with low affinity and high capacity seemed possible, but could not be verified for methodological reasons. In the light of the biochemical results and binding data, the wider range of ouabain concentration exerting a positive inotropic effect in the rat may be attributed to the existence in the latter of two populations of receptors with different affinities for ouabain and different capacities. In contrast, in the guinea-pig, there is a single population. Nevertheless it is probable that all the receptors in both species are part of the Na/K-ATPase complex and mediate a positive inotropic effect after ouabain-binding in an identical manner.