A clone of the regulator of G-protein signalling, RGS9, was isolated from a rat striatum-minus-cerebellum-minus-hippocampus subtracted library generated by directional tag polymerase chain reaction subtraction. The full-length cDNA clone encodes a 444 amino acid protein containing an 118 amino acid RGS domain, which corresponds to an evolutionarily conserved domain that is present in all members of the RGS family of proteins. Outside of the homology domain, RGS9 shows more extended similarity to human RGS6 and RGS7, rat RGS12, and the C. elegans protein EGL-10. During embryonic and early postnatal stages of development, two RGS9 transcripts of approximately 1.4 Kb and 1.8 Kb were detected in whole brain. After postnatal day 10, accumulation of the larger transcript increased progressively until adulthood at the expense of the smaller transcript, which was undetectable in the adult. In adult rat brain, the 1.8-Kb RGS9 transcript was detected in the striatum but not in other brain regions or peripheral tissues. In situ hybridization in rat and mouse demonstrates that RGS9 mRNA is expressed predominantly in medium-sized, spiny neurons of the neostriatum and in neurons of the nucleus accumbens and olfactory tubercle. Relatively strong signals were also detected in some hypothalamic nuclei. Its selective expression suggests that RGS9 may play an important role in modulation of the complex signalling pathways of the basal ganglia.