Reactive oxygen species (ROS) are produced and released into the extracellular spaces in numerous diseases and contribute to development and progression, for example, of inflammatory diseases, proteinuria, and tumor invasion. However, little is known about ROS-induced chemical changes of interstitial matrix proteins and their consequences for the integrity of the matrix meshwork. As basement membranes and other matrices are highly cross-linked and complex, the relatively simple matrix produced by Engelbreth-Holm-Swarm (EHS) sarcoma, and proteins isolated therefrom, were incubated in vitro with defined concentrations of ROS that were generated by the Fenton or xanthine oxidase/xanthine reactions. This resulted in two counter-current effects. Although up to approximately 15% of the EHS matrix proteins were released into the supernatant in a ROS dose-response relationship, the residual insoluble matrix was partially cross-linked by ROS. Matrix proteins released into the supernatants were examined by rotary shadowing, quantitative sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunoblotting, and fluorospectrometry for loss of tryptophans and formation of bityrosine residues. At relatively low ROS concentrations, selective liberation of morphologically intact laminin/entactin was found that, however, failed to reassociate and showed oxidative damage of its tryptophan residues. At higher ROS concentrations, laminin and entactin were progressively disintegrated, partially fragmented, and eventually completely degraded. At this point oligomers of type IV collagen predominated in the supernatant, and proteoglycans were not encountered at any concentration of ROS. Similar gradual molecular changes were also obtained when fractions of isolated soluble EHS matrix proteins were incubated with graded concentrations of ROS. In these experiments, the formation of covalently linked oligomers and aggregates paralleled the ROS-dependent formation of cross-linking bityrosine groups. ROS scavengers pinpointed to the hydroxyl radical as the most damaging radical species. Protease inhibitor experiments suggested that degradation of matrix proteins was caused primarily by the direct action of ROS and not by proteolysis by potentially contaminating proteases. Collectively, these results provide evidence that EHS matrix proteins show differential sensitivity to ROS-induced damage in a reproducible, sequential pattern, in the order entactin> laminin> type IV collagen, and that ROS cause partial dissociation and cross-linking of the EHS matrix.