‘Classic’ cardiotonic steroids (CTS, e.g., digoxin and ouabain) selectively inhibit Na+,K+-ATPase (the Na+ pump) and, via Na/Ca exchange, exert cardiotonic and vasotonic effects. CTS action is more complex than previously thought: Prolonged infusion of ouabain, but not digoxin, induces hypertension; in fact, digoxin antagonizes ouabain’s hypertensinogenic effect. We studied acute interactions between CTS in two indirect assays of Na+ pump function: myogenic tone (MT) in isolated, pressurized rat mesenteric small arteries, and Ca2+ signaling in primary cultured rat hippocampal neurons. The ‘classic’ CTS (0.3-10 nM) were ‘agonists’: they all increased MT70 (MT at 70 mm Hg) and augmented 3-4 μM glutamate-evoked Ca2+ (fura-2) signals. For example, 3-10 nM ouabain, digoxin and bufalin all increased myogenic constriction by 20-40% and neuronal Ca2+ signals by 80-200%. We then tested one CTS in the presence of another. Most CTS were either ouabain-like (ouabagenin, dihydroouabain, strophanthidin) or digoxin-like CTS (digoxigenin, digitoxin, bufalin). Within each group, the CTS were synergistic, but ouabain-like and digoxin-like CTS antagonized one another in both assays. For example, 3 nM ouabain-evoked increases in MT70 and neuronal Ca2+ signals were both reversibly attenuated by 65-75% by adding 10 nM digoxin or 10 nM bufalin. Conversely, 3 nM digoxin- or bufalin-evoked increases in MT70 and Ca2+ signals were attenuated by 65-75% by 10 nM ouabain, but augmentation by bufalin was not antagonized by digoxin and vice-versa. Rostafuroxin (PST2238, 5 μM), a digoxigenin derivative that displaces 3H-ouabain from Na+,K+-ATPase, and attenuates some forms of hypertension, antagonized ouabain's, but not digoxin's, effects. CTS bind to the α (catalytic) subunit of Na+ pump αβ protomers in a 1:1 ratio. Analysis of potential models suggests that, in vivo, Na+ pumps function as tetraprotomers [(αβ)4] in which the binding of a single CTS to one protomer blocks all pumping activity. The paradoxical ability of digoxin-like CTS to reactivate the ouabain-inhibited complex can be explained by de-oligomerization of the tetrameric state. The interactions between these common CTS may be of considerable therapeutic relevance.