The regional distribution of the serotonin uptake system was studied in rat brain using a specific polyclonal antibody raised against the putative extracellular loop between transmembrane domains 7 and 8 of the cloned rat serotonin transporter. Light microscope analysis with fluorescence and avidin-biotin-peroxidase techniques revealed a punctate staining as well as numerous labelled thin fibres, which exhibited accumulation of reaction end-product deposit over varicosities. These immunopositive processes were widely and heterogeneously distributed in the rat brain. High densities of immunoreactivity were seen within the caudate-putamen, amygdaloid complex, cortical areas, substantia nigra, ventral pallidum, Islands of Calleja, septal nuclei, interpeduncular nucleus, trigeminal motor nucleus and olfactory nuclei. We also found strong expression of serotonin transporter in the stratum oriens of area CA3 and, to a lesser extent, in the stratum oriens of CA1 and the stratum lacunosum molecular of CA1-CA3 regions of the hippocampus. Within the raphe nuclei, a moderate to high incidence of stained processes was observed, and immunopositive cell bodies were detected in the dorsal raphe nucleus. In addition, some immunoreactive fibres were present in the molecular and granular layers of the cerebellum as well as in the cochlear and olivary nuclei. In none of the regions analysed was evidence for glial staining obtained. The present immunocytochemical data reveal a widespread and heterogeneous distribution of the serotonin transporter in rat brain and suggest that serotoni transporter is preferentially sorted into axons, where it appears concentrated at varicosities and terminal boutons.