The effects of Na+ and Cl- on the binding of [3H]imipramine and the cocaine analog [125I]-beta-carbomethoxy-3 beta-(4-iodophenyl)tropane([125I]-beta-CIT) to the human platelet serotonin transporter have been measured. The ion dependence of beta-CIT binding is consistent with binding beta-CIT together with one Na+ ion, but not in an ordered sequence. Imipramine affinity, like beta-CIT affinity, is increased by Na+, but imipramine binding involves at least two Na+ ions. This conclusion is based on the observation that both imipramine association rate constants and equilibrium affinity constants show a sigmoidal Na+ dependence. As with beta-CIT, the imipramine and Na+ binding sequence is not strictly ordered. Cl- increases imipramine affinity, apparently by slowing dissociation. beta-CIT binding occurs even in the absence of Na+ and Cl-. This provided a means to measure substrate and inhibitor affinity in both the presence and absence of cotransported ions. Nontransported inhibitors, such as imipramine and citalopram, as well as the transport substrates serotonin and 3,4-(methylenedioxy)methamphetamine all displaced beta-CIT binding in the absence of NaCl. In the absence of Cl-, Na+ increased the affinity of nontransported inhibitors but not of substrates. The results suggest that Na+ and Cl- induce independent changes in the transporter binding site and that binding of substrates and inhibitors is affected differently by these changes.