Previous studies have shown that there is an increase in intracellular calcium concentration during hypoxia in the newborn and fetal brain. The high-affinity Ca++-ATPase enzyme, located on the outer membrane of the nucleus, has been shown to be a primary mechanism of Ca++ uptake into the nucleus in normoxia. In order to elucidate the mechanisms of regulation of nuclear calcium concentration during hypoxia, the present study investigates the effect of hypoxia on the activity of the high-affinity Ca++-ATPase. Nuclear membranes were prepared from the cortex of normoxic (n=5) and hypoxic(n=3) guinea pig fetuses at term. Tissue hypoxia was confirmed by a decrease in the levels of ATP and phosphocreatine. High-affinity Ca++ -ATPase activity was determined in the presence and absence of Mg++ in a medium containing 20 mM HEPES buffer (pH 7.0), 100 mM KCl, 95 μM CaCl2, 0 or 250 μM MgCl2, 1 mM ouabain, 1 mM ATP and 150 μg of nuclear membrane protein. The total, Mg++-dependent, and Mg++-independent Ca++-ATPase activities were calculated and expressed as nmoles/mg protein/hr. In the hypoxic group the total, Mg++ dependent, and Mg++-independent Ca++-ATPase activity increased compared to the normoxic group: 969.7 ± 79 vs 602.4 ± 90.9,p<0.005; 774.7 ± 147.4 vs 491.5 ± 93.8,p<0.01; and 193.8 ± 14.7 vs 132.8 ± 26,p<0.01. The results demonstrate that in the hypoxic nuclear membranes high-affinity Ca++-ATPase activity is increased, suggesting that the structure of the outer nuclear membrane is altered during hypoxia. Since the nuclear calcium signals control nuclear functions such as gene transcription and DNA synthesis, we speculate that the increased high-affinity Ca++-ATPase activity of the nuclear membrane, leading to an increase in nuclear Ca++ concentration, may be a mechanism of altered gene transcription in the hypoxic brain. (Funded by NIH # HD-20337).