Ro 11-2465 is a cyanide derivative of imipramine. In cerebral cortex homogenates, [3H] Ro 11-2465 displays a binding profile similar to that of [3H]imipramine. Agents compete with binding of [3H]Ro 11-2465 in an order of potency similar to their ability to block serotonin uptake, and raphe lesions greatly decrease the binding of [3H]Ro 11-2465. These observations suggest that the sites labeled by [3H]Ro 11-2465 are presynaptic. The binding of [3H]Ro 11-2465 is sodium ion-dependent, as are both the binding of [3H] imipramine and the serotonin uptake mechanism. In the presence of sodium ions, binding of [3H]Ro 11-2465 to brain tissue or platelets at 4 degrees is apparently irreversible. Binding is not displaced by high concentrations of the displacing agent desipramine or by repeated washing. However, by either removing sodium or increasing the assay temperature to 23 degrees, the ligand dissociates from the tissue. In tissue where [3H]Ro 11-2465 is irreversibly bound to receptor at 4 degrees, subsequent [3H]imipramine binding is decreased by about 50%. At temperatures greater than 23 degrees, [3H]Ro 11-2465 binding displays a temperature dependency similar to that of [3H]imipramine; that is, when temperatures are raised from 23 degrees to 30 degrees or 37 degrees there is no change in the Bmax, but the affinity of the ligand for the receptor is decreased. These data suggest that [3H]Ro 11-2465 binds to a discrete population of [3H]imipramine binding sites, comprising about one-half of the total [3H] imipramine binding sites.