Fibrin cross-linking by activated factor (F)XIII is essential for clot stability. In vitro, a common Leu34 polymorphism of the FXIII A-subunit increases the rate of thrombin-mediated FXIII activation, but not cross-linking activity upon complete FXIII activation. The effect of FXIII Val34Leu polymorphism on fibrin(ogen) cross-linking in vivo when vascular injury triggers the blood coagulation has not been studied yet. Using quantitative immunoblotting with antibodies raised against FXIII A-subunits, fibrinogen and γ-γ-dimers, the rates of FXIIIA cleavage and fibrin(ogen) cross-link formation in the fluid phase of 30-second blood samples collected at the site of microvascular injury were compared in the Leu34-positive and −negative healthy individuals and patients on long-term oral anticoagulation. In addition to accelerated FXIII activation, in healthy subjects the presence of FXIII Leu34 allele was associated with increased soluble γ-γ-dimer formation by 40% (1355±17 μg/L for Leu34 carriers vs 804.3±17 μg/L for Leu34 non-carriers; p=0.028) at the site of microvascular injury. This solution phase effect was abolished in coumadin-treated patients (369.4±75.9 μg/L for Leu34 carriers vs 290.5±35.9 μg/L for Leu34 non-carriers; p>0.05). The present study indicates that the Leu34 allele affects soluble γ-γ-dimer formation in untreated individuals, but not in those receiving acenocoumarol. Our data may help elucidate the impact of the FXIII Val34Leu polymorphism on fibrin crosslinking in vivo and its modulation by oral anticoagulants.