The proteolytic processing pattern of pro-ACTH/endorphin in rat hypothalamus is similar to the pattern in the pars intermedia; peptides the size of beta-endorphin, gamma-lipotropin (gamma-LPH), corticotropin-like intermediate lobe peptide (CLIP), alpha-melanotropin (gamma-MSH), joining peptide, and glycosylated gamma 3-MSH all represent predominant end products. Equimolar amounts of beta-endorphin-, alpha-MSH-, CLIP-, gamma-LPH-, and joining peptide-related immunoreactivity are found in hypothalamic extracts (approximately 3 pmol per hypothalamus). Although the proteolytic processing pattern in the hypothalamus is similar to that in the pars intermedia, a tissue-specific posttranslational processing pattern was detected. Ion-exchange analysis of beta-endorphin-sized immunoreactive material from hypothalamic extracts resolves three major forms, corresponding to beta-endorphin(1-31), beta-endorphin(1-27), and beta-endorphin(1-26). The alpha-N-acetylated forms of endorphin represent less than 10% of the total beta-endorphin immunoreactivity. Analyses of hypothalamic alpha-MSH-sized molecules with acetyl- and amide-directed alpha-MSH antisera suggest that hypothalamic alpha-MSH is fully amidated, but largely not alpha-N-acetylated. Fractionation by reverse-phase high-performance liquid chromatography (HPLC) confirms that greater than 85% of the alpha-MSH immunoreactivity corresponds to ACTH(1-13)NH2 or its sulfoxide, and less than 10% corresponds to alpha-MSH [alpha-N-acetyl-ACTH(1-13)NH2] or its sulfoxide. Isoelectric focusing demonstrates that 83-93% of hypothalamic CLIP is phosphorylated. Isoelectric focusing suggests that the majority of the hypothalamic gamma-LPH-sized immunoreactive material is indistinguishable from gamma-LPH synthesized by pituitary melanotropes. The minor extent of alpha-N-acetylation of alpha-MSH and beta-endorphin, the limited carboxyl-terminal proteolysis of beta-endorphin, and the extensive phosphorylation of CLIP represent major differences between the posttranslational processing patterns of pro-ACTH/endorphin in the hypothalamus and pars intermedia.