Proton pump inhibitors (PPIs) are used for the treatment of acid-related gastroduodenal disorders like ulcers and reflux esophagitis, but also for Helicobacter pylori (H.p.) eradication and prevention of nonsteroidal anti-inflammatory drug (NSAID) induced gastropathy. Their potent antisecretory effects are claimed to account for there efficacy in treatment of peptic ulcer disease. However, a comparison with other acid reducing agents suggests that PPIs also provide mucosal protection by mechanisms not yet fully understood. Therefore, the aim of this study was to investigate whether induction of the antioxidant enzymes heme oxygenase-1 (HO-1) and ferritin accounts for the gastroprotective effects of PPIs. All experiments have been performed using lansoprazole and omeprazole. Both PPIs induced the expression of the HO-1 gene in gastric epithelial cells as well as in murine macrophages and human endothelial cells. The observed increases of the HO-1 gene expression mediated by lansoprazole and omeprazole were independent of the proton pump inhibition. They occurred at transcriptional and translational levels and were associated with a stimulation of HO enzyme activity. Moreover, incubation of murine macrophages and human endothelial cells with omeprazole and lansoprazole led to an increase in ferritin protein expression in a concentration and time dependent manner. HO-1 mRNA induction was blocked by actinomycin D pointing to gene regulation at transcriptional level. Moreover both PPIs enhanced the HO-1 promoter activity in stable transfected murine fibroblastes. In addition, HO-1 mRNA induction in endothelial cells was abrogated in the presence of cycloheximide, suggesting involvement of protein de novo synthesis. Pre-treatment with superoxide dismutase did not affect PPI-mediated HO-1 induction precluding a mediator role of reactive oxygen species. There is evidence that the phosphatidylinositol-3-kinase (PI3K) has influence on the HO-1 induction by PPIs. Pre-incubation with the PI3K inhibitor LY294002 resulted in decreased HO-1 promoter activity and decreased HO-1 mRNA levels. HO-1 induction by PPIs was associated with a reduction of NADPH-dependent free radical formation after several hours of pre-treatment, pointing to an indirect antioxidant action. The role of HO-1 as a mediator of antioxidant activities was demonstrated by the direct radical scavenging effect of the HO-1 product bilirubin. In agreement with this, cell protection by omeprazole and lansoprazole was abrogated in the presence of the HO-inhibitor chromium mesoporphyrin IX, suggesting a causal link between PPI-mediated HO-1 induction and their radical scavenging effects. In summary, the present results demonstrate that PPIs activate antioxidant and anti-inflammatory pathways. These observations may explain, at least in part, the gastro protective effects of PPIs and may contribute to the beneficial activity of these drugs in patients with gastro-duodenal disease.