Guinea-pig ACTH has been found to be distinct from other mammalian ACTHs in having an alanine for proline substitution at position 24 and in having superagonist aldosterone-stimulating activity relative to synthetic ACTH(1–24) in an isolated rat glomerulosa cell bioassay. We have purified ACTH from extracts of guinea-pig anterior pituitary and confirmed its unusual structural characteristics by amino acid analysis and mass spectrometry. Using isolated rat adrenal fasciculata-reticularis and glomerulosa cell bioassays, guinea pig ACTH was found to have similar activity to that of human ACTH with respect to corticosterone- and aldosterone-stimulating activity, in terms of maximal steroid output but was slightly more potent in terms of the concentration which elicited half-maximal steroid secretion. Under the assay conditions used, guinea-pig ACTH appeared not to be a superagonist as previously suggested. Various biosynthetic derivatives of guinea-pig pro-opiomelanocortin were identified by amino acid analysis and mass spectrometry. Joining peptide, a major product of pro-opiomelanocortin processing, was found in extracts of both anterior and neurointermediate lobes of the pituitary. Post-translational modification of other products of intermediate lobe processing were observed. N-and O-acetylation of α-melanotropin, partial O-phosphorylation of corticotropin-like intermediate lobe peptide and carboxyl-terminal amidation of β-melanotropin were identified.