Elevated plasma fibrinogen levels are a major risk factor for thrombosis. This report shows two mechanisms by which fibrinogen can affect the fibrinolysis rate in vitro and thus may lead to thrombosis. First, the lysis rate of fibrin decreases as the initial concentration of fibrinogen increases. Second, a minor variant form of fibrinogen decreases the rate of fibrinolysis. This variant, gammaA/gamma' fibrinogen, has one altered gamma chain and is known to bind to factor XIII zymogen. In a fibrinolysis assay containing purified thrombin, fibrinogen, tissue-type plasminogen activator, and plasminogen, clots from gammaA/gammaA and gammaA/gamma' fibrinogen lysed at similar rates. However, when factor XIII was added, slower lysis was seen in gammaA/gamma' fibrin clots when compared with gammaA/gammaA fibrin clots. A D-dimer agglutination assay showed that the gammaA/gamma' clots were more highly cross-linked than the gammaA/gammaA clots. The lysis rates of gammaA/gamma' clots were similar to gammaA/gammaA clots in the presence of N-ethylmaleimide, a specific inhibitor of factor XIIIa. The gammaA/gamma' fibrin clots made in the presence of factor XIII showed increased proteolytic resistance to both plasmin and trypsin. Clots made from afibrinogenemic plasma reconstituted with gammaA/gamma' fibrinogen also showed significant resistance to lysis compared with gammaA/gammaA fibrinogen. These data demonstrate gammaA/gamma' fibrin is resistant to fibrinolysis, possibly as a result of concentrating factor XIII on the clot. The total fibrinogen concentration and the amount of gammaA/gamma' fibrinogen increase clot stability in vitro and thus may contribute independently to the risk of thrombosis in humans.