The neurointermediate lobe of the pituitary () contains the opioid peptides methionine enkephalin (MENK) and dynorphin 1–8 (DYN) in addition to oxytocin (OT) and vasopressin (AVP). If the opioids have a functional role, such as feedback control on OT or AVP release, the content or release of the opioids might be expected to change under conditions in which OT or AVP change. This expectation was examined by studying the synthesis, storage and release of the 4 peptides under conditions in which OT and AVP dynamics are known to be altered. Diestrus, diethylstilbesterol(DES)-treated, and day 22 pregnant rats were decapitated, the hypothalamo-neurohypophysial system (HNS) excised and either superfused in oxygenated Krebs buffer at 37 °C or stored at –80 °C for measurement of paraventricular and supraoptic nuclei mRNA content by in situ hybridization analysis. Peptide content of superfusates and homogenates were determined by specific RIAs. Compared with diestrus animals, DES treatment increased OT but decreased MENK. In term pregnant rats, OT, AVP, and DYN were increased over diestrus values, while MENK was again decreased. Release of the peptides from the isolated HNS paralleled changes in content. The hypothalamic mRNA for OTwas increased in DES-treated and pregnant rats while MENK mRNA was decreased. AVP and DYN mRNA was increased in pregnant animals. Although the was found to contain much more immunoreactive OT and AVP than MENK or DYN, under basal conditions the release of MENK was equal to or greater than the release of OT, while the release of DYN approached that of AVP. Stimulation with 100 mM potassium increased peptide release, but OT and AVP release was increased more than that of MENK or DYN, causing the release ratios of OT/MENK and AVP/DYN to increase 2- to 12-fold. These results indicate that DES treatment and pregnancy alter content and release of OT, AVP, MENK, and DYN by altering the hypothalamic synthesis of the peptides. If OT and MENK are colocalized, the finding that OT mRNA, content, and release was increased with estrogen-treatment and pregnancy, while that of MENK decreased, suggests that magnocellular neurons can differentially regulate colocalized peptides. The decrease in MENK mRNA, content, and release at term pregnancy suggests that the function of MENK is decreased at term, or, perhaps, during labor. The ratios of OT/MENK and AVP/DYN in the and that released during superfusion imply that each of these peptide pairs may exist in two separate pools.