SummaryBinding of ( 3 H)-ouabain to cultured cardiac muscle and non muscle cells from chicken embryos and neonatal rats has been characterized and correlated with ouabain-induced inhibition of the sodium pump, as well as with the positive inotropic action of the drug. 5. Our data demonstrate the existence of one single class of cardiac glycoside receptors in cultured cardiac muscle cells from chicken embryos. In rat heart muscle cells, inhibition of the sodium pump, as well as positive inotropy, is coupled to occupation of the low affinity, high capacity cardiac glycoside receptor. In contrast, occupation of the high affinity, low capacity ouabain binding site of rat heart cells does not produce any measurable inhibition of sodium pump activity nor positive inotropy. Nature and function of this high affinity site remain to be elucidated.