The guinea-pig has high levels of circulating cortisol. Though adrenocorticotropin (ACTH) levels are similar to those in other mammals, guinea-pig ACTH has been reported to have a single amino-acid substitution which results in increased bioactivity of the peptide. Pro-opiomelanocortin (POMC) is the precursor for ACTH, γ-melanocyte-stimulating hormone (γ-MSH) and the endogenous opioid peptide β-endorphin. Both to confirm this substitution in guinea-pig ACTH and to establish whether other non-conservative substitutions occur elsewhere in the precursor we cloned guinea-pig POMC. The guinea-pig alanine for proline substitution at position 24 of ACTH was confirmed. Potentially significant mutations were also identified in γ-MSH and β-endorphin. A similar pattern of POMC mRNA expression was obtained for guinea-pig and rat as determined by Northern analysis and in situ hybridization. Southern blot analysis indicated that guinea-pig POMC is a single-copy gene. Cloning and sequencing of guinea-pig POMC thus further demonstrate the divergence of the New World hystricomorph peptides from those in New World primates, and underscore the differences observed in other endocrine axes in the guinea-pig.