Heme oxygenase (HO) is the rate limiting enzyme in bilirubin production. The HO-1 isoform is inducible by various oxidant stresses including hyperoxia(1). In the metabolic reaction of HO, heme, a prooxidant is sequestered, bilirubin, an antioxidant is formed and ferritin is coinduced, thereby leading to sequestration of redox active iron(2). Thus the overall effect of HO may be to protect against oxidative stress. However, it is not clear whether HO is important in neonatal lung antioxidant defense. Therefore, we have chosen to investigate whether over-expression of HO enhances antioxidant defense in cultured rat fetal lung cells [RFL-6 cells (ATCC)]. Using the calcium phosphate reaction we transiently transfected RFL-6 cells with a PRC/CMV vector containing the full length HO-1 cDNA to obtain CMV promoter driven HO-1 expression. Confirmation of the vector map and inclusion of HO-1 cDNA was done by restriction analysis. HO-1 protein was assessed by immunohistochemistry and Western analysis,and HO-1 activity measured by gas chromatography. In separate experiments, transiently transfected RFL-6 cells were exposed to hyperoxia (95% O2/5% CO2) in a humidified chamber for 48 hours. We meassured cell viability by Trypan blue staining and clonogenic survival, LDH release, frequency of hyperoxia induced apoptosis, lipid peroxidation and protein oxidation and compared these values to untransfected control cultures exposed to air. HO-1 protein was increased 2 to 7 fold and HO activity increased 2 fold after transfection. Immnunohistochemistry revealed increased HO-1 signal in the perinuclear region of the cells. Exposure to 48 hours of hyperoxia was associated with increased cell viability (2 fold, n=3) and decreased protein oxidation (0.5 of control, n=3) but with increased frequency of apoptosis on day 2 of exposure. We conclude that significant overexpression of HO-1 can be achieved by the calcium phosphate reaction. Further investigation of HO-1 overexpression in resistance to hyperoxia may allow for novel therapeutic measures to enhance lung endogenous antioxidant defense.