The effect of the oral direct thrombin inhibitor ximelagatran and its active form, melagatran, on thrombin generation was investigated in vitro and ex vivo using a thrombin generation assay. In-vitro thrombin generation was triggered in human platelet-poor plasma by the addition of tissue factor, and the endogenous thrombin potential (ETP) was measured. The ETP IC(50) values for melagatran and the low-molecular-weight heparin dalteparin were 0.44 micromol/l and 0.06 IU/ml, respectively. In contrast to dalteparin, melagatran increased the time-to-thrombin peak in a concentration-dependent manner. ETP was also studied ex vivo in platelet-poor plasma collected from healthy male subjects (n = 54) at pre-dose and 2 h post-dose, with ximelagatran (60 mg) orally, dalteparin (120 IU/kg) subcutaneously, or control (water) orally. After ximelagatran or dalteparin administration, the time-to-thrombin peak was prolonged by 41 and 95%, and the ETP was decreased by 61 and 77%, respectively. Thus, melagatran, the active form of the oral direct thrombin inhibitor ximelagatran, efficiently delays and inhibits the generation of thrombin in plasma both in vitro and ex vivo.