Term fetal guinea pig brain Na + ,K + -ATPase has been characterized by its affinity for the steroid inhibitor ouabain, and has been found to exist in 2 distinct populations of high and low affinity. These low and high affinity sites also exist in adult brain; however, recently a very high affinity ouabain site has been found which is unique to fetal brain. The purpose of this study was to further characterize these very high affinity ouabain binding sites, and determine their sensitivity to hypoxia. Studies were performed on 6 fetuses obtained from pregnant guinea pigs at 60 days gestation (term). Fetuses in the normoxic group were delivered with the mother breathing room air. Fetuses in the hypoxic group were delivered after the pregnant dame had been in a 7% oxygen chamber for 60 minutes. After delivery of the fetuses in each group, the fetal brains were harvested, immediately frozen in liquid nitrogen, and stored at -80°C. Brain cell membranes were prepared, and ouabain binding studies were performed in a 0.25 ml reaction mixture containing 2 mM Tris-ATP, 100 mM NaCl, 2 mM MgCl 2 , 10 mM Tris buffer (pH 7.4), 50 mg brain cell membrane protein, and 3 H-ouabain (specific activity 21 Ci/mmol) varying from 1 to 200 nM. The binding reaction was carried out for 1 hr at 37°C, the samples filtered, and the radioactivity determined. Ouabain binding was determined in the normoxic and hypoxic samples in the presence and absence of erythrosin B (40 mM) a known inhibitor of high affinity ouabain binding sites. The normoxic brain preparation was found to have a Bmax of 84.2±13.6 pmol/mg protein and Kd of 24.6±4.5 nM, and in the presence of erythrosin B the Bmax was 5.9±3.8 pmol/mg protein (93.0% decrease) and Kd was 20.7±15.4 nM (p=NS). The hypoxic brain was found to have a Bmax of 74.7±8.3 pmol/mg protein and Kd of 22.9±1.9 nM, and with erythrosin B the Bmax was 7.1±3.9 pmol/mg protein (90.5% decrease) and Kd was 24.5±9.9 nM (p=NS). These results show that fetal brain has a unique very high affinity ouabain binding site which is resistent to hypoxia, and is sensitive to erythrosin B as are other high affinity binding sites. We speculate that the presence of a Na + ,K + -ATPase molecule with a very high affinity is essential for the early maturation of brain in this precocial species. The presence of such a Na + ,K + -ATPase molecule will be of an added advantage to the fetal brain under conditions such as hypoxia and ischemia so that energy is conserved in a low ATP environment.