Erythropoietin (Epo) synthesis is suppressed in normoxia and stimulated in hypoxia. To test the hypothesis that the cellular H2O2 level is important in the control of Epo synthesis, we have studied effects of modulators of H2O2 generation and degradation on Epo production in human hepatic cell cultures (hepatoma lines HepG2 and Hep3B). In addition, we measured the activities of antioxidant enzymes (catalase, superoxide dismutase, glutathione peroxidase) in cultures following hypoxia exposure or H2O2 treatment. The results show that the formation of immunoreactive Epo was stimulated in normoxic cultures by treatment with exogenous catalase thus mimicking the effect of hypoxia (24 h incubation periods). Epo production was also stimulated when scavengers of reactive O2 species (tetramethylthiourea, dihydrorhodamine) were added to the cells. On the other hand, stimulators of H2O2 generation (xanthine oxidase, glucose oxidase, NADH, NADPH) lowered Epo production in hypoxic cultures. Hypoxia exposure decreased superoxide dismutase activity and H2O2 treatment reduced catalase activity thus influencing the endogenous antioxidant defense system. These findings support the concept that reactive O2 species, primarily H2O2, act as messengers in the O2-dependent control of the hepatic production of Epo. Changes in the cellular activities of antioxidant enzymes appear to play only a minor role in this process.