TSG-6 protein (the secreted product of the tumor necrosis factor-stimulated gene-6), a hyaluronan-binding protein comprised mainly of a Link and CUB module arranged in a contiguous fashion, has been shown previously to be a potent inhibitor of neutrophil migration in an in vivo model of acute inflammation (Wisniewski, H. G., Hua, J. C., Poppers, D. M., Naime, D., Vilcek, J., and Cronstein, B. N. (1996) J. Immunol. 156, 1609-1615). It was hypothesized that this activity of TSG-6 was likely to be mediated by its potentiation of inter--inhibitor anti-plasmin activity (causing a down-regulation of the protease network), which was reliant on these proteins forming a stable, probably covalent ~120-kDa complex. Here we have shown that the recombinant Link module from human TSG-6 (Link_TSG6; expressed in Escherichia coli) has an inhibitory effect on neutrophil influx into zymosan A-stimulated murine air pouches, equivalent to that of full-length protein (which we produced in a Drosophila expression system). The active dose of 1 µg of Link_TSG6 per mouse (administered intravenously) also resulted in a significant reduction in the concentrations of various inflammatory mediators (i.e. tumor necrosis factor-, KC, and prostaglandin E2) in air pouch exudates. Link_TSG6, although unable to form a stable complex with inter--inhibitor (under conditions that promote maximum complex formation with the full-length protein), could potentiate its anti-plasmin activity. This demonstrates that formation of an ~120-kDa TSG-6·inter--inhibitor complex is not required for TSG-6 to enhance the serine protease inhibitory activity of inter--inhibitor. Six single-site Link_TSG6 mutants (with wild-type folds) were compared for their abilities to inhibit neutrophil migration in vivo, bind hyaluronan, and potentiate inter--inhibitor. These experiments indicate that all of the inhibitory activity of TSG-6 resides within the Link module domain, and that this anti-inflammatory property is not related to either its hyaluronan binding function or its potentiation of the anti-plasmin activity of inter--inhibitor.