Paroxetine (PAR) is a potent and selective inhibitor of serotonin uptake, and it has been demonstrated that 3H-PAR is a favorable candidate for labeling of the serotonin transport system. In this report, the binding of 3H-PAR to rat brain membranes was further investigated to obtain a better understanding of its role in the neuronal serotonin-uptake mechanism. Consistent with previous reports, it is indicated that 3H-PAR binds with high affinity to a site closely related to the serotonin-uptake mechanism. This binding is highly sodium dependent. The finding that depletion of brain serotonin content with p-chlorophenylalanine did not induce any alteration in the number and the affinity of the 3H-PAR-binding sites suggests the lack of serotonergic modulation on these sites. Repeated administration of desipramine, clomipramine, mianserin or deprenyl also failed to change 3H-PAR-binding sites in cerebral cortex and hippocampus. Such findings are compared to previous studies on the serotonin-uptake mechanism and the site labeled by 3H-imipramine, and discussed in respect to the biochemistry of affective disorders.