The thrombin generation potential is an in vitro measure for the capacity of an individual to generate thrombin and recognized as a reflection of a hypo- or hypercoagulable status. Measurement of the in vivo thrombin activity, however, may be of clinical significance. We evaluated a new assay for in vivo thrombin activity and compared it to the in vitro thrombin generation potential in patients with hemophilia A (N = 15), oral anticoagulation for atrial fibrillation (AF) (N = 20), subjects with active cancer (N = 21), and healthy volunteers (N = 10). Thrombin activity was measured with a commercially available oligonucleotide enzyme capture assay in argatroban-stabilized plasma samples. Thrombin generation potential was determined with a commercially available assay in citrated plasma. Thrombin activity was detected in 17 (30.4 %) patients (mean 0.30 mU/ml [SD 0.80]), and in 39 patients (69.6 %) no thrombin activity was present. In cancer patients, thrombin activity was detected in 11 patients (52 %) (range 0.14-5.00 mU/ml) and was particularly increased in 3 patients with vessel-invasive tumors (1.2, 1.5, and 5.0 mU/ml). In AF patients, thrombin activity was only measureable in two patients (10 %) (recent hematoma [0.4 mU/ml] and recent ischemic stroke [1.5 mU/ml]). Thrombin activity was detected in four patients (27 %) with hemophilia (range 0.29-1.75 mU/ml), all of whom had received a factor VIII infusion on the same day. Thrombin activity did not correlate with any of the parameters of the thrombin generation potential. Only patients in acute procoagulatory states or after clotting factor replacement had elevated in vivo thrombin activity, which was, however, unrelated to the in vitro thrombin generation potential.