Two approximately equal subpopulations of corticotropin-releasing hormone (CRH)-containing parvocellular axons can be identified in the external zone of the median eminence in normal (unadrenalectomized) rats: one that contains pro-vasopressin (AVP)-derived peptides (i.e. AVP, AVP-associated neurophysin and the carboxy terminal glycopeptide) copackaged with CRH in secretory vesicles, and another that contains no detectable pro-AVP-derived peptides. In this study, antibodies to pro-AVP-derived peptides were used to demonstrate for the first time that similar subpopulations of CRH-containing parvocellular perikarya exist in the paraventricular nucleus of the hypothalamus in normal rats treated with colchicine. Electron-microscopic immunocytochemistry was performed on serial ultrathin sections to identify neurosecretory cell perikarya containing CRH that also expressed pro-AVP peptides or pro-oxytocin-derived neurophysin. Of the CRH-positive neurons that were detected, more than half stained positively for two pro-AVP peptides: AVP-associated neurophysin and the carboxy-terminal glycopeptide. Many of these cells also stained for AVP, but staining was variable, making quantitation of AVP-positive cells difficult. The remaining CRH-positive neurons contained no detectable pro-AVP peptides, and less than 0.5% of these CRH perikarya contained oxytocin-associated neurophysin. In the neurons that stained positively for both CRH and the pro-AVP peptides, CRH and the pro-AVP peptides were localized in the same secretory vesicles. The pro-AVP expressing and pro-AVP-deficient CRH neurons were distributed differently within the paraventricular nucleus. The results demonstrate that the CRH neurosecretory system in normal rats is comprised of major AVP-expressing and AVP-deficient neuronal subpopulations, distinguishable not only on the basis of peptide phenotype but also by the different topographical distributions of their perikarya.