This report concerns ontogenetic aspects of the production and in vitro release of NH2-terminally acetylated forms of melanocyte-stimulating hormone (α-MSH) and β-endorphin by the pars intermedia of the pituitary gland of the mouse. In vitro biosynthetic analysis and radioimmunoassay revealed that approximately 12 h before birth most of the MSH in the fetal pars intermedia is present as des-Nα-acetyl α-MSH. The same non-acetylated peptide is at this stage also the major release form of melanotropin. In 1-day-old mice the level of α-MSH and diacetylated α-MSH had increased considerably, although des-Nα-acetyl α-MSH remained the major form. Five days after birth α-MSH and its diacetylated form constitute the major tissue and release form of the peptide, a situation very similar to that in adult mice. Acetylation of β-endorphin appeared to occur earlier in development, Nα-acetyl β-endorphin (1–31) being the major form of endorphin already in the fetal pars intermedia. It is concluded that in the mouse acetylation of melanotropin and acetylation of β-endorphin are not necessarily concomitant events. It could be established that the ability of the pars intermedia cells for cleaving Nα-acetyl β-endorphin (1–31) to yield C-terminally shortened forms of β-endorphin d