Studies have shown that calcium dependent activation of nuclear endonuclease causes a specific DNA fragmentation pattern characteristic of apoptosis. Non-specific DNA fragmentation can occur when activation of proteinase accompanies endonuclease activity. Hypoxia produces increased cellular calcium influx which may lead to DNA fragmentation and programmed cell death. This study investigates the pattern of DNA fragmentation following in utero hypoxia and recovery in the fetal guinea pig brain. Ten pregnant guinea pigs at term were studied. Brain tissue from fetuses of normoxic animals were compared to fetuses exposed to hypoxia but allowed to recover in utero 0, 24, 48, or 72 hours. Maternal hypoxia was induced by lowering inspired oxygen to 7% for 1 hour. Pregnant animals were subsequently allowed to recover in room air. Cerebral cortical neuronal nuclei were isolated and purified using a discontinious sucrose gradient. Genomic DNA was extracted and isolated. DNA (0.5 μg) was electrophoresed on a 1% agarose gel and stained with ethidium bromide. DNA base-pair fragments were compared to a standard DNA ladder of 100bp. Immediately following hypoxia specific DNA fragmentation was demonstrated producing lengths of 2000, 1500, 600, and 100 base-pair lengths. During recovery coexisting specific and random fragmentation of DNA was observed with an increasing random pattern demonstrated from 24 to 72 hours. This pattern of DNA fragmentation following hypoxia shows initial endonuclease activation which produces nucleosome-sized fragments which appears as specific bands in a “ladder” pattern. Over time, an increase in non-specific DNA fragmentation or“smear” pattern occurs during recovery. We conclude that DNA fragmentation immediately following hypoxia demonstrates endonuclease activity producing nuclear DNA damage. During recovery an increasing pattern of non-specific DNA fragmentation may represent increased proteinase and ongoing endonuclease activity of apoptosis.