The chronological appearances of three neuron systems, which contain peptides derived from proopiomelanocortin (POMC) and from preproenkephalin A, and amelanocyte-stimulating hormone (MSH), respectively, were examined in the hypothalamus of developing rats in situ and in grafts of the medial basal hypothalamus of 12.5-day-old embryos implanted into the third ventricle of adult female rats. POMC-cells labeled with anti-y-MSH, anti-adrenocorticotropin (ACTH), and anti,6-endorphin (End) antisera were the first to differentiate in the floor of the third ventricle on day 12.5-13.5 of gestation; subsequently they migrated to form the arcuate nucleus, and became to be labeled also with anti-a-MSH. At distinct later stages of development, two other neuron systems consisting of neurons staining exclusively with antisera against a-MSH, and against methionine-enkephalin-Arg6Gly7-Leug (Met-Enk-8) and Met-Enk, respectively, differentiated in discrete hypothalamic areas. These three kinds of neurons developed in grafts in the same way as in situ. It is concluded that the differentiation of neurons containing these different kinds of peptides are already determined on day 12.5 of gestation. Since the findings that adrenocorticotropin (ACTH), one of the anterior pituitary hormones, is processed from proopiomelanocortin (POMC) as one of its family peptides (23, 28), and that POMC is also present in the brain (16, 25, 38, 51), many anatomical, physiological, pharmacological and biochemical investigations have been made on POMC and its family peptides in the hypothalamus and pituitary (10, 11, 31, 48, 49). Because the POMC molecule includes the amino acid sequences of y-melanocyte-stimulating hormone (MSH), ACTH, a-MSH, ,8-lipotropin (LPH), ,8-endorphin (End), and methionineenkephalin (Met-Enk), it was originally believed that cells containing POMC are stained with the antisera against all these fragments. However, in the hypothalamus, although anti-ACTH, anti-B-End, anti-aMSH, and anti-,6’-LPH antisera stained the same neurons in the arcuate nucleus (2, 29, 43), anti-Met-Enk and anti-a-MSH antisera stained other separate neuron systems (3, 19, 37, 42), and Met-Enk was recently found to be derived enzymatically from a different precursor, preproenkephalin A (15, 30). Therefore, it seemed interesting to determine how the processing of POMC family peptides occurs in neurons during the ontogenetic process. Furthermore, it has been suggested that the manner of cleavage of POMC may be changed by some factors (41). To examine these problems,