The specific binding of [3H]imipramine to mouse brain membranes in an assay containing 120 mM NaCl and 5 mM KCl was similar in regional distribution and pharmacological specificity to that reported previously in rat and human brain. However, the absence of ions decreased the density of the specific binding of [3H]imipramine and did not affect the equilibrium dissociation constant. Sodium was the only cation, and halides were the only anions tested that enhanced the specific binding of [3H]imipramine. Chloride did not increase the density of binding in the absence of sodium. The ion-sensitive binding of [3H]imipramine was regionally dependent and was highly correlated with the uptake of 5-hydroxytryptamine (5-HT, serotonin) into synaptosomes from brain regions. 5-HT did not inhibit the binding of [3H]imipramine in the absence of ions. Antidepressants inhibited binding in the absence and presence of ions, but in the presence of ions inhibition curves were shifted to the left and the apparent complexity of inhibition was increased. Quantitative analysis of the inhibition of [3H]imipramine binding by antidepressants conducted in the presence of ions was consistent with two binding sites. Lesion of the serotonergic input to the cerebral cortex by 5,7-dihydroxytryptamine suggested that both the 5-HT-sensitive and ion-sensitive binding of [3H]imipramine were associated with serotonergic nerve terminals. [3H]Imipramine binding displaced by desipramine, but insensitive to 5-HT and ions, was not affected by the lesion. Thus, the binding of [3H]imipramine that is displaced by desipramine, the most common assay for [3H]imipramine binding, includes a component that is not associated with brain serotonergic nerve terminals and 5-HT uptake, and, in addition, a separable component that is highly correlated with serotonergic function. These data have important implications for studies of serotonergic neurons and for the interpretation of imipramine binding data.