Intermediate pituitary lobe cells from newborn rats were maintained in culture to determine the extent to which they continue to exhibit the tissue-specific properties of the newborn and adult intermediate pituitary lobes. At all times examined these cultures contained mostly .alpha.MSH-sized and corticotropin-like intermediate lobe peptide-sized peptides; the .alpha.MSH-sized peptides were predominantly diactetyl-ACTH-(1-13)NH2. After 6 days in culture, the intermediate pituitary lobe cells retained the ability to synthesize diacetyl-ACTH-(1-13)NH2. Compared to that of the adult, the newborn anterior pituitary lobe is enriched in high mol wt forms of ACTH-related molecules. Therefore, the ability of newborn anterior pituitary lobe cell cultures to develop the adult processing pattern in culture was investigated. After 6 days in culture, peptides the size of .alpha.MSH predominated rather than ACTH-(1-39), which is the major form found in the adult. Immunocytochemical studies showed that all cultured newborn corticotropes strongly stained for .alpha.MSH-related material. The .alpha.MSH-sized molecules were identified as ACTH-(1-13)NH2 by reverse phase HPLC. In 6-day-old cultures of neonatal anterior pituitary lobes grown in the presence of a synthetic glucocorticoid, dexamethasone, the amount of .alpha.MSH-sized material was diminished, and instead, precursor forms of the ACTH-related peptides were detected. In biosynthetic labeling experiments, the ratio of newly synthesized aCTH-(1-13)NH2 to ACTH-(1-39) was greatly reduced by treatment of the cells with dexamethasone. The extensive cleavage of ACTH-(1-39)and its regulation by dexamethasone are unique to newborn anterior pituitary lobe corticotropes; such plasticity is not observed in cultures of adult tissue.