Objective: A poorly characterized phagocyte-type NADPH oxidase, which is reportedly NADH- rather than NADPH-dependent, is a major source of endothelial reactive oxygen species (ROS) production. We investigated the molecular nature of this oxidase and the characteristics of NADPH- versus NADH-dependent O2− production in endothelial cells of three different species. Methods: NADPH oxidase expression in human, bovine and porcine endothelial cells was studied by RT-PCR and immunoblotting. O2− production was assessed by lucigenin chemiluminescence and cytochrome c reduction assay. Results: The NADPH oxidase subunits p47-phox, p67-phox, p22-phox, gp91-phox, and rac1 were all expressed in endothelial cells. NADPH-dependent O2− production by endothelial cells was readily detectable using lucigenin 5 μmol/l, was minimally affected by increasing lucigenin dose up to 400 μmol/l, and was abolished by diphenyleneiodonium. In contrast, NADH-dependent O2− production was only detectable with lucigenin ≥50 μmol/l, increased substantially with higher lucigenin dose, and was unaffected by diphenyleneiodonium. Predominance of NADPH- over NADH-dependent O2− production was confirmed in cell homogenates and by cytochrome c reduction assay. Conclusion: Endothelial cells express all components of a phagocyte-type NADPH oxidase. Like the neutrophil enzyme, the endothelial oxidase is preferentially NADPH- rather than NADH-dependent. NADH-dependent O2− production appears to be an artefact related to the use of lucigenin doses ≥50 μmol/l.