Previous studies have shown that the activation of Ca++/Mg++-dependent endonuclease that catalyzes the internucleosomal DNA cleavage is a biochemical marker of apoptotic cell death. The present study tests the hypothesis that brain tissue hypoxia results in increased activation of the Ca++/Mg++-dependent endonuclase leading to DNA fragmentation in the cerebral cortex of the guinea pig fetus. Pregnant guinea pigs of 58 days gestation were exposed to either 21% O2(normoxic n=6) or 7% O2 (hypoxic n=6) for 60 min. Fetal cortex was removed immediately after hypoxia and cerebral cortical nuclei were isolated using a discontinuous sucrose gradient. Brain tissue hypoxia in the fetus was documented biochemically by decreased levels of ATP and phosphocreatine. Ca++/Mg++-dependent endonuclease activation was determined by incubating cortical nuclei in the presence of 2 mM Ca++ and 5 mM Mg++. DNA was extracted and specific DNA fragments produced by the enzyme with 3'-OH ends were determined by random oligonucleotide primed synthesis (ROPS) assay. The ROPS assay was performed in a medium containing 10 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 7.5 mM DTT, 0.5 unit Klenow polymerase, 0.5 mM dTTP, 0.5 mM dATP, 0.5 mM dGTP, 1.65 mM dCTP, and 1uCi 3H-dCTP. The incorporation of 3HdCTP was determined. The results show that after calcium and magnesium activation, the 3HdCTP incorporation increased from 0.017 pmoles/ug DNA to 0.114 pmoles/ug DNA (6.6 fold) and from 0.014 pmoles/ug DNA to 0.237 pmoles/ug DNA (16.4 fold) in normoxic and hypoxic nuclei, respectively. The data indicate an increased activation of the Ca++/Mg++-dependent endonuclease in hypoxic nuclei. We speculate that during hypoxia an increase activity of Ca++/Mg++-dependent endonuclease will result in hydrolysis of phosphdiester bonds leading to DNA fragmentation and hypoxia-induced programmed cell death.