An endogenous inhibitor of Na(+)-K(+)-ATPase has been isolated from bovine hypothalamus and human plasma and structurally characterized as an isomer of the plant cardiac glycoside, ouabain (A. A. Tymiak, J. A. Norman, M. Bolgar, G. C. DiDonato, H. Lee, W. L. Parker, L.-C. Lo, N. Berova, K. Nakanishi, E. Haber, and G. T. Haupert, Jr. Proc.Natl.Acad.Sci. USA 90: 8189-8193, 1993; N. Zhao, L.-C. Lo, N. Berova, K. Nakanishi, J. H. Ludens, and G. T. Haupert, Jr. Biochemistry 34: 9893-9896, 1995). This hypothalamic inhibitory factor (HIF) acts on cardiovascular and renal tissues consistent with physiological regulation in vivo. Stimuli for the release of HIF from tissue are unknown. Hypoxia may be a stimulus for the elaboration of digitalis-like activity in humans, and high NaCl concentration in central nervous system stimulates ouabain-like activity in animals. We examined the ability of low O2 tension in vivo and in vitro to stimulate HIF release from midbrain and adrenal tissues in Wistar rats. In both tissues, hypoxia stimulated a remarkable release of an inhibitor cochromatographing with HIF, and this release was enhanced by 300 mM NaCl. Plasma from hypoxic rats also showed increased levels of the purified inhibitory activity. We conclude that hypoxia is a potent stimulus for the release of HIF or HIF-like activity and discuss the possibility that an Na(+)-K(+)-ATPase inhibitor could be involved in energy-conserving cellular adaptive responses to hypoxic or ischemic insult through ATP conservation.