Specific binding of [3H]imipramine to membrane preparation from rat cerebral cortex can be resolved in two distinct components: the high-affinity binding with a KD in nanomolar range (6.9 +/- 0.4 nM) and a maximum number of binding sites (Bmax) 285 +/- 19 fmol/mg protein and the low-affinity component with a KD of 292 +/- 45 nM and Bmax of 2459 +/- 428 fmol/mg protein. Tricyclic antidepressants, a non-tricyclic serotonin uptake inhibitor fluoxetine and a tetracyclic antidepressant maprotiline show a markedly different pattern in displacing [3H]imipramine binding at the high- and the low-affinity site. When the high-affinity sites were protected, the differences in potency of tested drugs in displacing specific [3H]imipramine binding, and the correlation with their ability to inhibit serotonin reuptake, were not observed. The Hill coefficients of competing drugs at low-affinity sites were markedly lower (0.45-0.69) and the shape of displacement curves indicated that more than one site may be involved in low affinity binding of [3H]imipramine.