Heme oxygenase (HO) catalyzes the rate-limiting enzymatic step of heme degradation and regulates the cellular heme content. Gene expression of the inducible isoform of HO, HO-1, is upregulated in response to various oxidative stress stimuli. To investigate the regulatory role of anoxia and reoxygenation (A/R) on hepatic HO-1 gene expression, primary cultures of rat hepatocytes were exposed after an anoxia of 4 hr to normal oxygen tension for various lengths of time. For comparison, gene expression of the noninducible HO isoform, HO-2, and that of the heat-shock protein 70 (HSP70) were determined. During reoxygenation, a marked increase of HO-1 and HSP70 steady-state mRNA levels was observed, whereas no alteration of HO-2 mRNA levels occurred. Corresponding to HO-1 mRNA, an increase of HO-1 protein expression was determined by Western blot analysis. The anoxia-dependent induction of HO-1 was prevented by pretreatment with the transcription inhibitor, actinomycin D, but not by the protein synthesis inhibitor, cycloheximide, suggesting a transcriptional regulatory mechanism. After exposure of hepatocytes to anoxia, the relative levels of oxidized glutathione increased within the first 40 min of reoxygenation. Pretreament of cell cultures with the antioxidant agents, beta-carotene and allopurinol, before exposure to A/R led to a marked decrease of HO-1 and HSP70 mRNA expression during reoxygenation. An even more pronounced reduction of mRNA expression was observed after exposure to desferrioxamine. Taken together, the data demonstrate that HO-1 gene expression in rat hepatocyte cultures after A/R is upregulated by a transcriptional mechanism that may be, in part, mediated via the generation of ROS and the glutathione system.