Amiodarone has been shown to affect cell membrane physicochemical properties, and it may produce a state of cellular hypothyroidism. Because the sarcolemmal Na(+)-K+ pump is sensitive to changes in cell membrane properties and thyroid status, we examined whether amiodarone affected Na(+)-K+ pump function. We measured Na(+)-K+ pump current (Ip) using the whole-cell patch-clamp technique in single ventricular myocytes isolated from rabbits. Chronic treatment with oral amiodarone for 4 weeks reduced i.p. when myocytes were dialyzed with patch-pipettes containing either 10 mM Na+ or 80 mM Na+. In myocytes from untreated rabbits, acute exposure to amiodarone in vitro reduced i.p. when patch pipettes contained 10 mM Na+ but had no effect on i.p. at 80 mM Na+. Amiodarone had no effect on the voltage dependence of the pump or the affinity of the pump for extracellular K+ either after chronic treatment or during acute exposure. We conclude that chronic amiodarone treatment reduces overall Na(+)-K+ pump capacity in cardiac ventricular myocytes. In contrast, acute exposure of myocytes to amiodarone reduces the apparent Na+ affinity of the Na(+)-K+ pump. An amiodarone-induced inhibition of the hyperpolarizing Na(+)-K+ pump current may contribute to the action potential prolongation observed during treatment with this drug.