The binding of [3H] digoxin to purified canine cardiac sarcolemmal vesicles was characterized. Scatchard analysis of saturation isotherms yielded linear plots with a maximal binding capacity of 174+/-31.9 pmol/mg, a dissociation constant of 31.7+/-4.59 n M, and a Hill coefficient of 0.947+/-0.02 (mean+/-SEM), suggesting that [3H] digoxin bound to a single class of sites. In contrast to their marked effect on steady-state serum digoxin levels when administered in combination, quinidine, verapamil, and amiodarone were without effect on equilibrium binding of [3H] digoxin. Thus, increased steady-state serum concentrations of digoxin resulting from combination therapy with these particular drugs probably will have cardiac effects that may increase the risk of digitoxicity to the patient.