The distribution of imipramine binding sites in rat brain was examined by the LKB film method of autoradiography. Brain sections were labeled in vitro with [3H]imipramine in the presence or absence of 100 microM unlabeled desmethylimipramine to define non-specific binding. The binding of [3H]imipramine to brain sections was saturable, specific and of a high affinity (2-6 nM). There were very high levels of imipramine binding sites in the dorsal and medial raphe nuclei and over the superficial layers of the superior colliculus. Moderately high concentrations were observed in the olfactory tubercule, interpeduncular nucleus and substantia nigra, among others. There were low levels of binding sites over white matter and in the ventral thalamus. The distribution of imipramine binding sites parallels the distribution of serotonin terminals in rat brain. These results are consistent with the hypothesis that the high affinity imipramine binding site is associated with the presynaptic uptake site for serotonin.