Manganese superoxide dismutase (SOD2) is a nuclear encoded and mitochondria localized antioxidant enzyme that converts mitochondria derived superoxide to hydrogen peroxide. This study investigates the hypothesis that mitochondria derived reactive oxygen species (ROS) regulate ionizing radiation (IR) induced transformation in normal cells. Mouse embryonic fibroblasts (MEFs) with wild type SOD2 (+/+), heterozygous SOD2 (+/-), and homozygous SOD2 (-/-) genotypes were irradiated with equitoxic doses of IR, and assayed for transformation frequency, cellular redox environment, DNA damage, and cell cycle checkpoint activation. Transformation frequency increased ( approximately 5-fold) in SOD2 (-/-) compared to SOD2 (+/+) MEFs. Cellular redox environment (GSH, GSSG, DHE and DCFH-oxidation) did not show any significant change within 24 h post-IR. However, a significant increase in cellular ROS levels was observed at 72 h post-IR in SOD2 (-/-) compared to SOD2 (+/+) MEFs, which was consistent with an increase in GSSG in SOD2 (-/-) MEFs. Late ROS accumulation was associated with an increase in micronuclei frequency in SOD2 (-/-) MEFs. Exit from G(2) was accelerated in irradiated SOD2 (+/-) and SOD2 (-/-) compared to SOD2 (+/+) MEFs. These results support the hypothesis that SOD2 activity and mitochondria generated ROS regulate IR induced transformation in mouse embryonic fibroblasts.