We have developed potent and selective thrombin inhibitors with a novel non-peptidic structure. A bicyclic lactam was used as the scaffold on which various P1 and P3 motifs were substituted. Herein, we report the in vitro and in vivo properties of four representatives of this novel class of inhibitors. Their Ki values were less than 10 nM, they inhibited equally both free and clot-bound thrombin, and they displayed high level of specificity for thrombin over other serine proteases (trypsin, factor Xa, activated Protein C, and plasmin). They prolonged the clotting time of human plasma to twice the control value in coagulation assays (TT, APTT, and PT) at a concentration below 3 microM. Their anticoagulant activities using rat plasma were similar to, although slightly weaker, than with human plasma. Furthermore, they inhibited thrombin-induced platelet aggregation (human and rat) at concentrations close to their Ki values for thrombin. These molecules demonstrated similar dose response antithrombotic efficacy in rat arterial and venous thrombosis models when given as i.v. bolus followed by infusion. Antithrombotic efficacy of 85% and greater was observed at a dose of 5-7 microM/kg/hour in each model. Bicyclic lactam inhibitor 3, at a dose which caused a complete inhibition of visible thrombus formation in the venous and arterial models of thrombosis, showed a 1.9-2.1 and a 4.0-4.8-fold shift in APTT and TT, respectively. Unfortunately, the bicyclic lactam inhibitors exhibited low oral bioavailability in rats. Therefore, this novel class of bicyclic lactam thrombin inhibitor has the potential to be promising intravenous antithrombotic agents for the treatment of arterial as well as venous thrombosis and warrants further investigation.