Previous studies have shown that nuclear calcium signals control a variety of nuclear functions including gene transcription, DNA synthesis, DNA repair and nuclear envelope breakdown. We have previously demonstrated in cerebral nuclei of the guinea pig fetus that high-affinity Ca++ -ATPase enzyme activity increases under hypoxic conditions. The present study aims to evaluate kinetic changes in Ca++ -ATPase activity during hypoxia. Pregnant term guinea pigs were exposed to an FIO2 of 0.21 or 0.07 for 1 hour. Nuclear membranes were prepared from the cortex of 5 control and 4 hypoxic guinea pig fetuses. Tissue hypoxia was confirmed by a decrease in the levels of ATP and phosphocreatine. High-affinity Ca++-ATPase activity was determined in a medium containing 20 mM HEPES buffer (pH 7.0), 100 mM KCl, 95 μM CaCl2 (free calcium = 2.49 μM), 250 μM MgCl2, 1mM ouabain, 1 mM ATP and 150 μg of nuclear membrane protein. Ca++-ATPase activity was determed and expressed as nmoles/mg protein/hr. Lineweaver Burke plots were created and maximal enzyme velocity(Vmax) and dissociation constant (Ka) for Ca++ and ATP were determined in the presence of free Ca++ concentrations ranging from 0-3.95 mM, and Na-ATP concentrations from 0-2.5 nm, respectively. As previously shown Ca++-ATPase activity increased significantly during hypoxia compared to the normoxic group. Enzyme Vmax for Ca++ was 540 ± 109 nmol Pi/mg protein/h for C and 735 ± 79 nmol Pi/mg protein/h for H(p<0.05). The Ka for Ca++ was 0.33 ± 0.22 μM for C and decreased to 0.11 ± 0.07 μM for H (p<0.05). Enzyme Vmax for ATP was 1949 ± 350 nmol Pi/mg protein/h for C and 2167 ± 276 nmol Pi/mg protein/h for H (p=NS). Similarly the Ka for ATP was 70.7 ± 9.6 nM for C and 76 ± 22.3 nM for H (p=NS). The data demonstrate that the hypoxic enzyme has increased affinity for its activator Ca++, but its affinity for ATP is unchanged. We speculate that brain tissue hypoxia modifies the active sites of the high affinity Ca++-ATPase enzyme. Since nuclear calcium signals control nuclear functions such as gene transcription and DNA synthesis, the increased high-affinity Ca++-ATPase activity of the nuclear membrane may lead to an increase in nuclear Ca++ concentration and alter gene transcription in the hypoxic brain.