Previous studies have shown that the mRNA encoding the Na+/Cl(-)-dependent "orphan" transporter Rxt1 is expressed exclusively in the central nervous system (CNS). In the present study, specific antibodies were raised in rabbits for the detailed mapping of this transporter in the rat. The C-terminal part of Rxt1 was fused with glutathione-S-transferase (Rxt1ct-GST) and the resulting fusion protein was used as antigen. The specificity of the antiserum toward Rxt1 was confirmed by immunofluorescent, Western blot, and immunoautoradiographic experiments. In cerebral cortex membranes, Rxt1-like material recognized by the antiserum is a glycosylated protein of 97-116 kDa. This protein was the most abundant in the caudate-putamen, followed, in decreasing order, by the cerebral cortex approximately hippocampus > cerebellum > brainstem > spinal cord. In contrast, no immunoreactive material could be detected in peripheral tissues (tongue, thymus, heart, lung, spleen, kidney, adrenals, liver, skeletal muscle, intestine, testis). Immunoautoradiographic labeling with affinity-purified anti-Rxt1ct-GST antibodies showed high levels of Rxt1-like material in the olfactory bulb, cerebral cortex, striatal complex, hippocampal formation, superior layer of the anterior colliculus, cortex, and deep nuclei in the cerebellum. The regional distribution of Rxt1-like material generally matched that of GABAergic and glutamatergic projections in agreement with previous in situ hybridization data.