Anchorage-dependent cells in culture attach initially to proteins adsorbed to the culture substrate from the medium, and produce and deposit a subcellular matrix during the course of the cultivation. The aim of this study was to determine whether the concentration of O(2) in the culture atmosphere affects the accumulation of type IV collagen and laminin under human endothelial-cell monolayers. Enzyme-linked immunoassays on decellularized polystyrene substrates showed less type IV collagen, but not less laminin, under cells incubated in the standard atmosphere (5% CO(2) in air, i.e., approximately 20% O(2)) compared to an atmosphere of 5% O(2) and 5% CO(2) in N(2). Type IV collagen accumulation was inhibited via oxidative stress, because the inhibitory effect of 20% O(2) was antagonized by antioxidant ascorbic acid, and mimicked by prooxidant pyrogallol and exogenous H(2)O(2). Measurements of endogenous H(2)O(2) accumulation demonstrated that endothelial cells partially adapt to the high O(2) concentration. These results may have implications in endothelium modeling in vitro and in engineering of endothelial cell sheets and endothelialized vascular grafts.