Macrophage migration has been extensively studied in vitro on artificial substrates but in vivo macrophages migrate through connective tissue and fibrin gel meshworks that comprise the stroma of many inflammatory reactions and solid tumors. Studies were therefore undertaken to investigate macrophage migration in this more biologically relevant matrix cast either with or without nitrocellulose filter support. Macrophage migration in fibrin gels depended on both fibrin and thrombin concentrations and on the nature of fibrin crosslinking. With gamma-chain crosslinking only, macrophage migration was enhanced as compared with untreated filters at fibrin concentrations of up to 5 mg/ml. However, when fibrin alpha-chains were also crosslinked, as occurs in vivo, macrophage migration was inhibited at approximately 3 mg/ml fibrin and was stopped altogether at 5 mg/ml. Thrombin concentrations of 1 unit/ml favored maximal macrophage migration. Depletion of contaminating fibronectin did not affect these results. The pore size of fibrin gels is well below the minimum that permits macrophage migration through nitrocellulose filters. Thus, to penetrate fibrin gels, macrophages could enlarge effective pore size by local fibrinolysis or by pushing apart fibrin strands. If fibrinolysis is responsible, this process must be highly localized because only small amounts of fibrinolysis accompanied extensive macrophage migration in fibrin gels; moreover, fibrinolysis inhibitors did not affect macrophage migration. We conclude that the fibrin deposited in inflammatory reactions and solid tumors is present in forms and concentrations that either facilitate or inhibit macrophage migration; fibrin may therefore regulate macrophage participation in these and other pathologic reactions.