The binding of [3H]cyanoimipramine to serotonin uptake sites in rat brain slices was studied using quantitative autoradiography. Binding was of high affinity and was to a single class of binding site. This is in contrast to results previously obtained by others with [3H]imipramine where two binding sites were observed. The sites labeled by [3H]cyanoimipramine had properties consistent with this ligand labeling serotonin uptake sites, as: (1) binding is displaced by drugs which are potent inhibitors of serotonin uptake but not by drugs which are weak inhibitors of uptake; (2) binding is dependent on the presence of sodium ions as is the uptake of serotonin; (3) binding is almost completely eliminated in the brains of rats lesioned by the serotonin neurotoxin 5,7-dihydroxytryptamine; (4) the distribution of binding sites throughout the rat brain is highly correlated with that found previously for [3H]indalpine, a potent serotonin uptake inhibitor, and for [3H]imipramine. The properties of binding of [3H]cyanoimipramine make it an ideal ligand for the quantitative autoradiography of serotonin uptake sites.