Using an in situ hybridization screen for cDNA clones of brain region-specific mRNAs, we isolated a rat transcript that encodes a ribosomal protein S27. Searching GenBank DNA databases, we found two S27 protein isoforms. One isoform, encoded by multiple genes, is extant in archaea and eukarya, but not bacteria. The second isoform appears to be recently evolved because it has been identified only in mammals. Multiple transcripts encode each isoform and exhibit different tissue expression patterns throughout rat brain and periphery, with abundant expression in the hypothalamus. In situ hybridization studies revealed predominant expression of S27(1) in distinct hypothalamic nuclei, such as the paraventricular, supraoptic, suprachiasmatic, arcuate, and circularis nuclei, whereas expression of S27(2) mRNA was discretely expressed in select neurons of the periventricular and supraoptic nuclei. Combined with the genetic evidence that S27 has extraribosomal functions in plants, the complexity of S27 biology observed here may suggest auxiliary functions for S27 proteins in the mammalian nervous system.