Oxygen modulates the glucagon-dependent activation of the phosphoenolpyruvate carboxykinase (PCK) gene. The respiratory chain or heme proteins have been proposed to function as O2-sensors. The functions of the respiratory chain are impaired by uncouplers such as 2,4-dinitrophenol (DNP); those of ferro-heme proteins are affected by carbon monoxide (CO), which locks heme in the oxy conformation. Therefore, the effects of different concentrations of CO and DNP on the glucagon-dependent induction of PCK mRNA and PCK activity were investigated at different physiological oxygen tensions in primary rat hepatocyte cultures. The cells were cultured under standard conditions from 4-24 h. After addition of fresh media PCK was induced with 1 nM glucagon. PCK mRNA and PCK activity were elevated after 2h and 3h, respectively, to 100% at 16% O2 (mimicking arterial oxygen tensions) and to about 60% at 8% O2 (mimicking venous oxygen tensions). CO counteracted the reduced induction at lower oxygen tensions: Under 8% O2 + 2% CO PCK mRNA could be elevated again to about 90% and PCK activity to about 80%. CO did not impair the induction by insulin of ornithine decarboxylase (ODC) and the incorporation of 14C-leucine into total protein. CO did not cause lactate dehydrogenase (LDH) to leak from the cells or influence the cell structures at the microscopical level. DNP (50 microM) unspecifically lowered PCK gene expression without affecting its modulation by oxygen. These results are in line with the proposal that a ferro-heme protein rather than the respiratory chain acted as an O2 sensor in the activation of the PCK gene.