Microdialysis in conjunction with a highly sensitive radioimmunoassay was used to monitor the in vivo release of arginine vasopressin (AVP) within the supraoptic nucleus (SON) of the rat brain (n = 70). Thirty-min dialysates were collected in urethane-anesthetized animals before, during and after hypertonic or hypotonic pulses were delivered via the probe. As compared to artificial cerebrospinal fluid (aCSF)-perfused controls, 1M aCSF given over a period of 210 min resulted in an increased intranuclear AVP release which, however, reached its peak only in the post-stimulation period, i.e. after replacement of hypertonic with isotonic aCSF again (rebound phenomenon). Hypertonic (0.5 M, 1 M or 2 M) pulses given 150 min after the first (1 M) pulse resulted in 3 different rebound responses: a marked decrease (to 25.3%, P less than 0.001), no change or slight increase (132%, n.s.). As shown by the response to correction of the hypertonicity to normal as well as by perfusion of hypotonic aCSF (0.01 M), release of AVP within the SON appears more responsive to a reduction than to an elevation in the osmolality of aCSF. Omission of Ca2+ from and addition of EGTA to the aCSF decreased the osmotically stimulated, but not the basal AVP release. If K(+)-hypertonic aCSF was used, however, basal AVP levels increased significantly; in contrast, the rebound increase failed to differ from aCSF-perfused controls. It is concluded from these findings that release of AVP from intact neuronal structures in the SON is responsive to changes in osmolality with an apparent greater sensitivity to decrease in osmolality.