The binding of cocaine, d-amphetamine and dopamine to the site on the dopamine transporter labeled by [3H]mazindol was investigated in rat striatal membranes. N-Ethylmaleimide inhibited about 95% of the specific binding of 5 nM [3H]mazindol in a concentration-dependent manner. The effect of 10 mM N-ethylmaleimide was completely prevented by cocaine (EC50 of 3 microM), but neither 300 microM dopamine nor d-amphetamine afforded any significant protection. On the other hand, high concentrations of cocaine, d-amphetamine and dopamine provided similar protection against inhibition by 0.1 mM N-ethylmaleimide. Taken together these data support the hypothesis that a significant portion of the cocaine binding domain on the transporter is distinct from that of either dopamine or amphetamine. This distinction may be sufficient to allow properly designed drugs to prevent cocaine binding without inhibiting DA transport.