PCR differential display screening has recently identified a rat mRNA termed CART (cocaine- and amphetamine-regulated transcript) which is transcriptionally regulated in the striatum following acute administration of psychomotor stimulants. The endogenous CART transcript is expressed in diverse rat brain structures, as well as endocrine tissues. The deduced CART protein contains a hydrophobic signal sequence, suggesting that it may be targeted for secretion. Thus, the CART protein may represent a novel neuroendocrine signaling molecule. The study described here represents a complete analysis of the human CART cDNA and gene. The complete nucleotide (nt) sequence of the approx. 900-nt human CART transcript is contained within three distinct exons, with the entire human CART gene localized to a segment of genomic DNA approx. 2 kb in length. The human CART cDNA sequence is 80% identical to the corresponding rat cDNA, with 92% homology observed within the deduced protein-coding region. Third-nt changes account for most of the latter differences, with CART exhibiting 95% identity between these two species at the amino-acid sequence level. PCR/Southern blot analysis of DNA isolated from human/rodent somatic cell hybrid panels localizes the CART gene to human chromosome 5. Lastly, Northern blot analysis reveals that the gross pattern of distribution of CART mRNA in human brain is similar to that previously observed in rat. These overall similarities suggest that CART plays a conserved role within the mammalian neuroendocrine system.