Recently, we have shown that mammalian plasma cross-reacts with an antibody to a bufodienolide Na/K ATPase inhibitor, marinobufagenin. In rat aorta, marinobufagenin induced vasoconstriction via inhibition of the vascular smooth muscle Na/K pump, whereas ouabain had its predominant effect on pumps localized to nerve endings. Na/K ATPase inhibitory effects of ouabain and marinobufagenin were studied in two membrane fractions isolated from Fisher 344xBN rat thoracic aorta by sucrose density gradient centrifugation. One fraction contained predominantly the alpha-3 isoform of Na/K ATPase and represented membranes from the perivascular nerve endings (neuronal plasmalemma). The other membrane fraction, containing predominantly the alpha-1 isoform, was derived from the vascular smooth muscle sarcolemma. The IC50 for inhibition of the Na/K ATPase by ouabain and marinobufagenin were 2.6 nmol/L and 0.14 micromol/L in the neuronal plasmalemma, and 50 nmol/L and 2.1 nmol/L in sarcolemma, respectively. These results confirm the view that differential responsiveness to endogenous digitalis-like factors is a functional feature of alpha isoforms of Na/K ATPase.