The present study examines the interaction of Na+ and K+ with the binding of the cocaine analogue 3beta-(4-[125I]iodophenyl)tropane-2beta-carboxylic acid isopropyl ester to dopamine transporters (DATs) in rat striatal synaptosomal membranes at 37 degrees C. The binding increases with [Na+] from 10 to 100 mM and decreases with higher [Na+]. The presence of K+ reduces the maximal stimulatory effect of Na+ and causes a nonlinear EC50 shift for Na+. K+ strongly inhibits the binding at low [Na+]. Increasing [Na+] produces a linear IC50 shift for K+. Saturation analysis indicates a single binding site changing its affinity for the radioligand depending on [K+]/[Na+] ratio in the assay buffer. A reduced Bmax was observed in the presence of 10 mM Na+ and 30 mM K+. Both high [Na+] and high [K+] accelerate the dissociation of the binding, and K+-induced acceleration was abolished by increasing [Na+]. Least squares model fitting of equilibrium data and kinetic analysis of dissociation rates reveal competitive interactions between Na+ and K+ at two sites allosterically linked on the DAT: One site mediates the stimulatory effect of Na+, and the other site involves the radioligand binding and the inhibitory effect of cations on the binding. Various uptake blockers and substrates, dopamine in particular, display reduced potency in inhibiting the binding at a higher [K+]/[Na+] ratio.