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 nM, 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.