Cortistatin is a 14-residue putative neuropeptide with strong structural similarity to somatostatin and is expressed predominantly in cortical GABAergic interneurons of rats. Administration of cortistatin into the brain ventricles specifically enhances slow-wave sleep, presumably by antagonizing the effects of acetylcholine on cortical excitability. Here we report the identification of cDNAs corresponding to mouse and human preprocortistatin and the mRNA distribution and gene mapping of mouse cortistatin. Analysis of the nucleotide and predicted amino acid sequences from rat and mouse reveals that the 14 C-terminal residues of preprocortistatin, which make up the sequence that is most similar to somatostatin, are conserved between species. Lack of conservation of other dibasic amino acid residues whose cleavage by prohormone convertases would give rise to additional peptides suggests that cortistatin-14 is the only active peptide derived from the precursor. As in the rat, mouse preprocortistatin mRNA is present in GABAergic interneurons in the cerebral cortex and hippocampus. The preprocortistatin gene maps to mouse chromosome 4, in a region showing conserved synteny with human 1p36. The human putative cortistatin peptide has an arginine for lysine substitution, compared to the rat and mouse products, and is N-terminally extended by 3 amino acids.