The quaternary amine, bretylium, is a class III antiarrhythmic drug used to treat ventricular tachycardia and fibrillation. The primary mode of action for bretylium is thought to be inhibition of voltage-gated K(+) channels. While the Na,K-ATPase has been the pharmacological target of cardiac glycosides for over a century, recent evidence has shown that bretylium may also inhibit the Na pump. Our experimental findings support and extend these previous reports and provide definitive evidence supporting the previous suggestion that bretylium and K compete for the Na pump. We find that bretylium inhibits the Na pump in a dose-dependent manner in both Na,K-ATPase (IC(50) 4.5 mM) and Rb flux experiments (IC(50) 3.5 mM). Furthermore, we show that bretylium and Rb(+) competes for an extracellular site by measuring ouabain-sensitive (86)Rb flux in intact human red blood cells; that is, there is an apparent increase in K(m) for Rb(+) in the presence of 5 mM bretylium, while V(max) remains unchanged. We also determined that unlike K(+), bretylium does not facilitate the hydrolysis of E2-P. However, it stabilizes this conformation by reducing the ability of K(+) to facilitate dephosphorylation. Finally, we show that bretylium, like K(+), reduces [(3)H]ouabain binding to the Na pump. Taken together, these data are consistent with bretylium binding to the extracellular facing cation site within the E2-P state of the enzyme. Moreover, these findings suggest that bretylium may serve as an effective tool for freezing the pump in an extracellularly cation-bound phosphorylated intermediate, which will aid in future structural analyses.