Varying results on the effect of factor XIII (FXIII) Val34Leu on venous thrombotic risk have been reported. The probability of a true association between this polymorphism and venous thrombotic risk would be enhanced by a laboratory phenotype associated with this polymorphism and with the thrombotic risk. The aim of this study was to assess the effect of FXIII Val34Leu, FXIII activity and subunit levels on venous thrombotic risk in a large case-control study, The Leiden Thrombophilia study (LETS). We found higher FXIII activity for 34Leu carriers (Leu/Leu: 158.0, Val/Val: 95.0). FXIII subunit levels were not associated with genotype. Higher FXIII activity was associated with a slightly decreased thrombotic risk [Odds ratio (OR): 0.8, 95% confidence intervals (CI): 0.5-1.3]. This effect was not present for elevated FXIII subunit levels. Higher FXIII activity was also associated with a higher dissociation index (percentage A2B2 complex dissociated after activation by thrombin for a fixed time interval). This index was higher for FXIII 34Leu carriers. The risk of deep venous thrombosis was slightly decreased for carriers of the 34Leu allele [OR: 0.9 (95%CI: 0.7-1.1)]. For homozygous 34Leu carriers the OR was 0.7 (95%CI: 0.4-1.3). This finding, suggesting a weak protective effect, was completely restricted to men. An overall estimate of thrombotic risk was calculated by using earlier reports on the risk of FXIII Val34Leu. The overall risk estimate for homozygous 34Leu carriers was 0.8 (95%CI: 0.6-1.0). In this study, a weak protective effect against venous thrombosis was found, of FXIII 34Leu as well as of increased FXIII activity.