Evidence of factor XI (FXI) activation in vivo is scarce. In addition, it remains uncertain whether thrombin, factor XIIa (FXIIa), or perhaps another protease is responsible for FXI conversion. We investigated the activation of FXI in eight healthy volunteers after infusion of a low dose of endotoxin (4 ng/kg of body weight). Activation of prekallikrein FXII, FXI, and prothrombin was measured with sensitive enzyme-linked immunosorbent assays (ELISAs), and FXI activation was measured with a novel enzyme capture assay that detects noncomplexed FXIa. Activation of FXI was apparent with a significant plasma peak level of noncomplexed FXIa of 10 to 11 pmol/L at 1 and 2 hours after endotoxin infusion, followed by a gradual increase in FXIa-FXIa inhibitor complexes, measured in the ELISAs, with a summit of 11 to 15 pmol/L at 6 and 24 hours, respectively. In accordance with previous studies, thrombin generation was detected 1 hour after endotoxin infusion to become maximal after 3 to 4 hours. In contrast, we did not find any evidence of contact activation, because markers of activation of prekallikrein and FXII remained undetectable. From the FXIa data a theoretical model was constructed which suggested that inhibition of FXIa does not take place in the plasma compartment, but is localized on a surface. These data provide the first evidence for FXI activation in low-grade endotoxemia and suggest that FXI is activated independently of FXII.