Tricyclic and nontricyclic serotonin [5-hydroxytryptamine (5-HT)] uptake inhibitors are widely used for the treatment of depression. Here, we show that both the tricyclic antidepressant imipramine and the nontricyclic antidepressant citalopram competitively inhibit 5-HT transport mediated by the recombinant rat 5-HT transporter SERT1. For citalopram, the concentration producing half-maximal transport inhibition was in the same order of magnitude as its K(D) value determined by equilibrium binding. In contrast, the inhibitory potency of imipramine was more than one order of magnitude lower than its K(D) value. Our data are consistent with low-affinity imipramine binding occurring at or close to the substrate recognition site, which also binds citalopram. Occupation of the high-affinity imipramine binding site on SERT1 did not affect 5-HT transport but allosterically displaced citalopram from the substrate recognition site. Consequently, low concentrations of imipramine partially protected 5-HT transport from citalopram inhibition. This protection was only observed in the presence of Na+ because high-affinity imipramine binding is strictly sodium-dependent. Thus, depending on which of its binding sites on SERT1 is occupied, imipramine may exert distinct effects on 5-HT uptake mediated by the recombinant rat 5-HT transporter.