Extracts of the medial basal hypothalamus (MBH), the preoptic anterior hypothalamus (POA), or the pituitary gland of young (4-month-old) and old (18-month-old) female rats were fractionated on columns of Sephadex G-75 superfine. Five forms of immunoreactive corticotropin (ACTHi) were found in the MBH or POA: greater than 40K, 30-40K, 20-30K, 5.7K, and 4.5K. In contrast, 4 forms of ACTHi were found in the pituitary gland: 30-40K, 20-30K, 5.7K, and 4.5K. Thus, hypothalamic tissue contains a large form of ACTH (greater than 40K ACTH) which is not present in the pituitary gland. We tentatively identified greater than 40K ACTH as a large form of pro-opiocortin, 30-40K ACTH as pro-opiocortin, 20-30K ACTH as ACTH biosynthetic intermediate, 5.7K ACTH as glycosylated ACTH 1-39, and 4.5K ACTH as ACTH 1-39. The content of ACTHi in the MBH and POA of old rats was lower than that of young rats. Nevertheless, regardless of the age of the animals, the fractional amount of 30-40K ACTHi was high in the MBH (a region that includes the presumed site of biosynthesis of pro-opiocortin) compared to that in the POA (a region that is distant to the site of biosynthesis of pro-opiocortin). Moreover, the reduced fractional amount of 30-40K ACTHi in the POA was associated with an increased fractional amount of greater than 40K, 20-30K, 5.7K, and 4.5K ACTHi. These findings are consistent with a precursor-product relationship between the 30-40K ACTHi and 20-30K ACTHi, 5.7K ACTHi, and 4.5K ACTHi.(ABSTRACT TRUNCATED AT 250 WORDS)