Polymorphonuclear leukocytes have been implicated in connective tissue injury in a variety of disease processes. To gain insight into mechanisms by which neutrophils might degrade connective tissue macromolecules in the presence of proteinase inhibitors, we have used a model system that allows neutrophils to be held in vitro under physiologic conditions in close proximity to a very proteinase-sensitive substrate, ¹²⁵I-labeled fibronectin. We have found: (a) neutrophils spread rapidly on the fibronectin substrate; (b) fibronectin proteolysis by neutrophils is largely attributable to released elastase, and is linearly related to cell number over the range of 2,000 to 30,000 cells per assay; (c) oxidants released from neutrophils stimulated by opsonized zymosan or phorbol myristate acetate do not protect released elastase from inhibition by α₁-proteinase inhibitor or α₂-macroglobulin; (d) neutrophil myeloperoxidase and enzymatically generated superoxide anion render α₁-proteinase inhibitor ineffective against fibronectin proteolysis when neutrophils are added 30 min later; and (e) α₁-proteinase inhibitor and α₂-macroglobulin incompletely inhibit fibronectin proteolysis by neutrophils (79.8±6.3 and 73.5±12.0%, respectively.) The data suggested that proteolysis due to neutrophils that are in contact with susceptible macromolecules may occur due to partial exclusion of inhibitors from the cell-substrate interface. Although confirming that α₁-proteinase inhibitor is ineffective against neutrophil-derived proteolysis after exposure to oxidants, these studies did not support the hypothesis that oxidants released from stimulated neutrophils enhance activity of proteinases they release in the presence of α₁-proteinase inhibitor. We anticipate that further studies with this test system will be helpful in defining conditions that modulate inflammatory connective tissue injury in diseases such as pulmonary emphysema and rheumatoid arthritis.