Morphine withdrawal results in serious affective and somatic symptoms including activation of the hypothalamo-pituitary-adrenocortical (HPA) axis. To reveal secretory, activational and transcriptional changes in the hypothalamus of morphine-dependent rats during naloxone precipitated opioid withdrawal, we measured corticosterone secretion, c-Fos induction and heteronuclear (hn)RNA levels of corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) in naïve and morphine dependent animals injected with saline or 5 mg/kg naloxone. Naloxone precipitated morphine withdrawal resulted in a significant increase in corticosterone secretion and induction of neuronal activation in the hypothalamic paraventricular nucleus (PVH) 2 h after challenge. Using probes complementary to intronic sequences of genes encoding neuropeptides in parvocellular neurosecretory neurons of the PVH, we found robust increases in CRH and AVP hnRNAs in morphine dependent rats during naloxone precipitated withdrawal. Naïve rats and animals that were implanted with morphine pellets for 8 days did not display significant up-regulation of ongoing neuropeptide expression in the parvocellular compartment of the PVH. In addition to hypophyseotropic neurons, naloxone precipitated withdrawal resulted in a marked activation in autonomic-related projection neurons in PVH and in the magnocellular neurons in the PVH and supraoptic nuclei. These activations however were not associated with induction of CRH or AVP hnRNAs.