The production of alpha-melanotropin (alpha-N-acetyl-ACTH(1-13)NH2 (alpha MSH) in rat intermediate pituitary was investigated using reverse phase high performance liquid chromatography, immunoassays, and biosynthetic labeling. Extracts of rat intermediate pituitary contain primarily alpha-N,O-diacetyl-ACTH(1-13)NH2, with smaller amounts of des- and monoacetyl-ACTH(1-13)NH2. No significant amount of ACTH(1-14)OH- or ACTH(1-13)OH-related material was observed. Analysis of the newly synthesized alpha MSH-sized peptides produced by freshly prepared rat intermediate pituitary cell suspensions revealed labeled peptides co-migrating primarily with des-, mono-, and diacetyl-ACTH(1-13)NH2. In contrast, when extracts of 2-week-old intermediate pituitary cultures were analyzed, the major peak of material detected with the general NH2-terminal ACTH antiserum eluted at the position expected for alpha-N,O-diacetyl-ACTH(1-14)OH; smaller amounts of material co-migrating with des- and monoacetyl-ACTH(1-14)OH were also detected. Analysis of the newly synthesized alpha MSH-sized peptides produced by 16-day-old cultures of intermediate pituitary cells revealed labeled peptides co-migrating with des-, mono-, and diacetyl-ACTH(1-14)OH. Thus, rat intermediate pituitary cells in culture maintain the ability to perform several major proteolytic cleavages of pro-ACTH/endorphin as well as the ability to alpha-N-acetylate alpha MSH and beta-endorphin, but selectively lose the ability to alpha-amidate the carboxyl terminus of alpha MSH.