In the rat hypothalamus, proenkephalin (PE) mRNA synthetizing cells were detected by in situ hybridization, using synthetic oligodeoxy-nucleotides, from embryonic day 14 (E14) in the presumptive anterior hypothalamic area (AHA) and preoptic part of the bed nucleus of the stria terminalis (BST), and from E18 in the developing median preoptic area, perifornical area, suprachiasmatic nucleus, dorsomedial and ventromedial hypothalamic nuclei. In the paraventricular nucleus, cells expressed PE gene in the late prenatal stages; both parvo- and magnocellular neurons synthetized PEmRNA in the early postnatal stages. Cells expressing PE gene were observed after birth in the lateral preoptic area, lateral hypothalamus, medial and lateral parts of the BST. PEmRNA was also found from E14 in the striatum, from E18 in the central and medial amygdaloid nuclei, the medial group of the thalamic nuclei, and postnatally in a second more anterior structure of the thalamus. In the hypothalamus, a clear similarity was observed between adult and developmental distributions of PE gene expressing cells. The early onset of PE gene expression in the developing rat diencephalon suggests an involvement of PE in developmental processes, such as cell proliferation and differentiation; the presence of PE during the perinatal period may also indicate the appearance of adult neural regulations.