The effect of the cardiotonic aglycone, strophanthidin, on sodium and potassium efflux, membrane potential, membrane conductance, potassium permeability, and the shape of the action potential of the giant axon of the squid, Loligo pealei, was examined. Strophanthidin depolarized the membrane to an extent determined by the intracellular sodium concentration, except in axons pretreated with cyanide, in which the effect is abolished. Cyanide itself hyperpolarized the axon membrane. Axons treated with strophanthidin appear to be better potassium electrodes, but this observation is fully accounted for by the stimulating effect of [K]o on an electrogenic sodium pump. The increase in potassium efflux produced by strophanthidin is also well accounted for by the observed membrane depolarization and the known dependence of potassium permeability on membrane potential (e-fold increase in efflux per 6.4 mV depolarization). Strophanthidin has no demonstrable effect on membrane conductance apart from that due to the observed depolarization. These findings support the view that cardiotonic steroids, at least in nerve, are specific inhibitors of the sodium pump, devoid of effects on permeability that might interfere with the study of electrogenic pumping. The alteration in the shape of the action potential after exposure to strophanthidin (deepening of the "underswing") suggests that the strophanthidin-induced membrane depolarization results from the inhibition of a true electrogenic pump, and not from ion redistributions in the vicinity of the membrane.