Plasma fibronectin (Fn) can both enhance phagocytic clearance of microparticulate debris by macrophages as well as incorporate it into the lung extracellular matrix (ECM). The goal of this study was to document that N-ethylmaleimide (NEM)-treated human plasma Fn (HFn) would lose its ability to incorporate into the lung ECM in vivo even though it would retain its ability to stimulate test particle phagocytosis and bind to fibrin. Using dual-label immunofluorescence, we compared the lung deposition of purified normal HFn and NEM-alkylated HFn (NEM-HFn) after their intravenous injection into postoperative nonbacteremic and bacteremic sheep in relationship to the localization of endogenous sheep Fn. Two days after a sterile surgical thoracotomy, sheep were infused with either 5 × 10⁸ Pseudomonas aeruginosa (postsurgical bacteremic model) or the diluent (nonbacteremic model). They also received a bolus 100-mg injection (5 min) of either HFn or NEM-HFn. Analysis of serial lung biopsies harvested at 2-h intervals demonstrated little deposition of NEM-HFn compared with HFn in the lung interstitial matrix of postoperative nonbacteremic sheep. In contrast, enhanced deposition of both HFn and NEM-HFn was observed in the lungs of postoperative bacteremic sheep. However, in the lungs of bacteremic sheep, HFn displayed a diffuse fibrillar deposition pattern in the lung characteristic of ECM incorporation, whereas the enhanced NEM-HFn deposition, especially in the interstitial ECM region of the lung, was primarily focal and punctate, with very little fibrillar incorporation. Immunofluorescent analysis with antibodies specific to fibrinogen, Fn, and lung macrophage surface antigens coupled with immunoperoxidase staining for HFn antigen revealed that the punctate fluorescence pattern was due to both the binding of HFn to fibrin and its colocalization with inflammatory cells. Thus treatment of plasma Fn with low concentrations of NEM will limit its normal in vivo fibrillar incorporation into the interstitial ECM region of the lung.