Macrophages are important players of the immune system to fight infections since they eliminate microorganisms. During this process they often encounter a wide range of oxygen concentrations, from roughly 13% O2 (PO2 = 760 x 0.13 mmHg) in arterial blood to less than 1% O2 in inflamed tissue. Macrophages contribute to the elimination of microorganisms by releasing oxygen derivates such as hydrogen peroxide. The objective of this study was to test macrophage activity under various O2 and H2O2 concentrations such as present during infection. We exposed macrophages to steady-state O2 concentrations between 21% and 1% O2 and steady-state H2O2 concentrations between 0 and 20 M using a novel enzymatic system. The system uses glucose oxidase (Gox) and catalase (Cat) in standard, open-system, cell culture vessels. Macrophage activity was determined as change in phagocytosis and as release of antimicrobial H2O2 into extracellular medium. We show that O2 concentrations below 7% enhance the activity of macrophages of the THP-1 cell line in a dose-dependent way; with doubled activity at 1% O2 compared to 21% O2 conditions. We further show that macrophages are able to function in an environment of high H2O2concentrations and are even stimulated by H2O2 concentrations below 20 M. The activity of hypoxic macrophages is up to 3-fold enhanced in the presence of H2O2 as compared to the activity triggered by low O2 conditions alone. Our data show that hypoxia and H2O2, as present in infectious conditions, strongly enhance macrophage activity. The data further demonstrate the usefulness and versatility of the Gox/Cat system for studies of infectious and inflammatory diseases.