The immunoreactive (IR) human N-terminal (hNT) of pro-opiomelanocortin (POMC) was measured by specific radioimmunoassay (RIA) and characterized by molecular sieving chromatography, concanavalin A-affinity chromatography and reversed-phase high-performance liquid chromatography (HPLC). The IR hNT levels were 380 +/- 144 ng/g wet wt (mean +/- SD) in the adrenal medulla (N:6), 21.6 +/- 6 ng/g wet wt in the adrenal cortex (N:6), and 45.6% ng/g wet wt in pheochromocytoma tissues (N:3). The IR hNT content of the adrenal medulla was found to be at least twice as high as that of the IR ACTH on a molar basis. Molecular sieving chromatography of IR hNT and IR gamma-3-melanotropin (MSH) showed two major molecular forms (apparent molecular weights of 14 and 12 kilodalton). These major forms were also separable using reversed-phase HPLC. In addition, a part of the IR ACTH material from the adrenal medulla extracts was eluted with an apparent molecular weight of 12 kilodalton. This latter form of IR ACTH was also separated from authentic human ACTH (1-39) by HPLC. Results obtained from concanavalin A-agarose chromatography suggest that one part of the IR gamma-3-MSH material from the adrenal medulla might be non-glycosylated. These results indicate the presence of IR hNT and IR gamma-3-MSH-like material in the human adrenal and also suggest a different processing pathway for POMC from that in the pituitary gland.