Products of the proenkephalin gene are not only neurotransmitters but may also influence brain development. The ontogeny of the expression of the proenkephalin gene in neocortex was studied in embryonic and postnatal rats with in situ hybridization. At embryonic day 14, the proliferating cells in the ventricular zone strongly expressed the gene. Thereafter, the expression decreased and was hardly detectable up to embryonic day 21. At the day of birth and during the subsequent week, proliferating cells in the subventricular zone were labelled. The expression of the proenkephalin gene in proliferating neuronal and glial progenitors indicates that gene products may affect proliferation and/or commitment. In the neocortex, cells which strongly expressed the gene were first seen at postnatal day 7 in the outer part of the neocortex. Seven days later, a second band of positive cells had appeared in the inner part of the cortex, i.e. the adult pattern of distribution had been established. Thus, in rat neocortex the expression of the proenkephalin gene developed in an outside-first, inside-last mode.