We observed coexistence of corticosteroid-binding globulin (CBG) with vasopressin (VP) and oxytocin (OT) in magnocellular neurons in rat hypothalamus by combined immunoperoxidase staining and immunofluorescence. A portion of the supraoptic and of the paraventricular neurons showed double immunostaining of CBG with either VP or with OT. CBG staining was intensified by pretreating animals with colchicine to block axonal transport. CBG was also observed in widespread axonal projections throughout the lateral hypothalamus, the median eminence and the posterior pituitary lobe. Single ependymal cells and some of the endocrine cells in the anterior lobe contained specific CBG immunoreactivity. IN SITU hybridization of semithin sections with a synthetic oligonucleotide probe to CBG mRNA provided staining of magnocellular hypothalamic neurons, but not ependymal cells or anterior lobe cells. Western blots of CBG extracted by affinity chromatography from hypothalamus homogenates showed a band at approximately 50 kDa. Our observations indicate the intrinsic expression of CBG in peptidergic hypothalamus neurons in rat. The multiple locations of CBG-expressing neurons indicate multiple functional properties, probably exceeding the role of a mere steroid transporter. CBG is likely to be subject to axonal transport and secretion in a neuropeptide-like fashion, perhaps involved in neuroendocrine regulation, which may include stress responses. Corticosteroid-binding globulin circulates in plasma and binds its cognate ligand with high affinity, offering a steroid delivery system to target tissues by a variety of mechanisms. This study shows that CBG plasma levels (in normal subjects) peak in the early afternoon. This leads to attenuation of the diurnal free cortisol level rhythm compared to total cortisol. Measurement of CBG concentration in plasma and the total CORT level gives opportunity to determine free CORT (and therefore biological active) concentration.