1. The influence of amiodarone on [3H]ouabain (OUA) binding to myocardial Na+, K(+)-ATPase was studied at various KCl concentrations in guinea pig heart microsomal preparations to test the hypothesis that the drug acts on the same receptor sites as cardiac glycosides. 2. First, a series of assays for OUA binding to Na+, K(+)-ATPase were performed in the range of 64-800 nM at 2.5, 5.0 and 10.0 mM K+ concentration. The drug exhibited increasing binding tendency with increasing concentrations and elevation in K+ levels. 3. Competitive binding assays were then performed at 256, 512 and 800 nM OUA in the presence of 50, 100 and 200 microM amiodarone at 5.0 mM KCl, respectively. At each OUA concentration, a concentration-dependent left-to-right shift was observed in the binding affinity with increasing amiodarone concentration. similar assays at 2.5 and 10.0 mM K+ showed the same trends. These effects were significant for 200 mM amiodarone at all K+ levels and OUA concentrations. 4. Furthermore, different OUA concentrations were also shown to displace amiodarone in a concentration-dependent fashion. 5. These results indicate that amiodarone competes with OUA for specific binding sites on myocardial microsomal Na+, K(+)-ATPase. They lead to the conclusion that myocardial Na+, K(+)-ATPase is a possible receptor for some of the cardiac actions of amiodarone, such as its proarrhythmic effects.