Heme proteins acting as oxidases which produce H2O2 have been proposed to function as O2 sensors. In order to find out whether the modulation by O2 of PCK gene activation and the stimulation of the ALD A gene by venous O2 operate via H2O2, the effects of different concentrations of H2O2 and catalase as H2O2 scavenger were studied in rat hepatocyte cultures under different O2 tensions. Primary hepatocytes were treated with 0.1 nM glucagon, 50 microM H2O2 and/or 100 micrograms/ml catalase each at arterial O2 or venous pO2. PCK mRNA was induced by glucagon maximally under arterial O2 and only half maximally under venous O2. ALD A mRNA was induced only by venous O2. H2O2 enhanced the induction of PCK mRNA to similar levels under venous O2 tensions and the induction of ALD A mRNA under both O2 was completely inhibited. Addition of catalase antagonized the actions of H2O2 completely. These findings support the hypothesis that an H2O2-generating heme protein is involved in the O2 sensing system regulating gluconeogenic and glycolytic gene expression in response to O2.