It was found that ouabain and marinobufagenin, specific inhibitors of Na+,K+-ATPase, increased the contraction of the isolated rat diaphragm by ∼15% (positive inotropic effect) at EC50 = 1.2 ± 0.3 nM and 0.3 ± 0.1 nM, respectively, which was indicative of the participation of the ouabain-sensitive Na+,K+-ATPase α2 isoform. Analysis of the dose-response curves for the effect of ouabain on the resting membrane potential of muscle fibers in the absence and in the presence of 100 nM acetylcholine (hyperpolarizing the membrane) showed the presence of two pools of Na+,K+-ATPase α2 that differed in affinity for ouabain. Only the high-affinity pool (IC50 ∼ 9 nM) mediates the hyperpolarizing effect of nanomolar concentrations of acetylcholine. Most likely, it is this pool of that is involved in the positive inotropic effect of ouabain, which can be a mechanism of regulation of the muscle function by circulating endogenous inhibitors of Na+,K+-ATPase.