Plasminogen plays an integral role in the inflammatory response, and this participation is likely to depend on its interaction with cell surfaces. It has previously been reported that isolation of human neutrophils from blood leads to a spontaneous increase in their plasminogen-binding capacity, and the basis for this up-regulation has been explored as a model for mechanisms for modulation of plasminogen receptor expression. Freshly isolated human peripheral blood neutrophils exhibited relatively low plasminogen binding, but when cultured for 20 hours, they increased this capacity dramatically, up to 50-fold. This increase was abolished by soybean trypsin inhibitor and was susceptible to carboxypeptidase B treatment, implicating proteolysis and exposure of carboxy-terminal lysines in the enhanced interaction. In support of this hypothesis, treatment of neutrophils with elastase, cathepsin G, or plasmin increased their plasminogen binding, and specific inhibitors of elastase and cathepsin G suppressed the up-regulation that occurred during neutrophil culture. When neutrophils were stimulated with phorbol ester, their plasminogen binding increased rapidly, but this increase was insensitive to the protease inhibitors. These results indicate that plasminogen binding to neutrophils can be up-regulated by 2 distinct pathways. A major pathway with the propensity to markedly up-regulate plasminogen binding depends upon the proteolytic remodeling of the cell surface. In response to thioglycollate, neutrophils recruited into the peritoneum of mice were shown to bind more plasminogen than those in peripheral blood, suggesting that modulation of plasminogen binding by these or other pathways may also occur in vivo.