The distribution and sites of synthesis in rat CNS of NTT4, a novel orphan member of the Na+/Cl(-)-dependent neurotransmitter transporter family, were determined by immunohistochemistry and hybridization histochemistry. Antibodies raised against recombinant fusion proteins, corresponding to residues of NTT4, and 35S-labelled oligodeoxyribonucleotide probes, were used to delineate the cellular distribution of the transporter at the protein and mRNA levels. High levels of immunoreactivity (mainly in the neuropil) were found in the olfactory bulb, cerebral cortex, striatum, hippocampus, thalamus, substantia nigra, pontine nuclei, cerebellum and spinal cord. The lowest levels were associated with the lateral hypothalamic area and deep mesencephalic nuclei. In situ hybridization signals correlated well with the immunoreactivity, and demonstrated a widespread distribution of NTT4 transcripts exclusively in neurons. NTT4 transcripts appeared widely codistributed with the N-methyl-D-aspartate receptor subunit 1 (1-4b), i.e. spliced variants characterized by a common 5' 63 bp insertion. These results indicate that the transporter was associated with neuronal processes in specific glutamate innervated CNS regions. Although the substrate transported by NTT4 remains unknown, our findings suggest a possible role for this carrier protein in glutamate/glycine neurotransmission.