Alpha-melanocyte-stimulating hormone (α-MSH) is a neuropeptide expressed in pituitary and brain that is known to regulate energy balance, appetite control, and neuroimmune functions. The biosynthesis of α-MSH requires proteolytic processing of the proopiomelanocortin (POMC) precursor. Therefore, this study investigated the in vivo role of the prohormone convertase 2 (PC2) processing enzyme for production of α-MSH in PC2-deficient mice. Specific detection of α-MSH utilized radioimmunoassay (RIA) that does not crossreact with the POMC precursor, and which does not crossreact with other adrenocorticotropin hormone (ACTH) and β-endorphin peptide products derived from POMC. α-MSH in PC2-deficient mice was essentially obliterated in pituitary, hypothalamus, cortex, and other brain regions (collectively), compared to wild-type controls. These results demonstrate the critical requirement of PC2 for the production of α-MSH. The absence of α-MSH was accompanied by accumulation of ACTH, ACTH-containing imtermediates, and POMC precursor. ACTH was increased in pituitary and hypothalamus of PC2-deficient mice, evaluated by RIA and reversed-phase high pressure liquid chromatography (RP-HPLC). Accumulation of ACTH demonstrates its role as a PC2 substrate that can be converted for α-MSH production. Further analyses of POMC-derived intermediates in pituitary, conducted by denaturing western blot conditions, showed accumulation of ACTH-containing intermediates in pituitaries of PC2-deficient mice, which implicate participation of such intermediates as PC2 substrates. Moreover, accumulation of POMC was observed in PC2-deficient mice by western blots with anti-ACTH and anti-β-endorphin. In addition, increased β-endorphin1−31 was observed in pituitary and hypothalamus of PC2-deficient mice, suggesting β-endorphin1−31 as a substrate for PC2 in these tissues. Overall, these studies demonstrated that the PC2 processing enzyme is critical for the in vivo production of α-MSH in pituitary and brain.