Aeruginosins are a family of naturally occurring oligopeptides that share a common bicyclic amino acid core structure. Many compounds in the family are inhibitors of serine proteases, such as thrombin and trypsin. Thrombin is an important enzyme in the blood coagulation cascade, and is a promising target for anticoagulant drug development. In order to understand the structure-activity relationship (SAR) and to find selective thrombin inhibitors, we synthesized a series of aeruginosin 298-A analogs, in which the P(2) bicyclic amino acid was replaced by a L-proline residue. The structure optimization was focused on modification of the P(1) position. In choosing the P(1) group, an effort was made to avoid using the highly basic guanidine groups present in nearly all naturally occurring aeruginosins. The synthesis and enzyme assays of these aeruginosin analogs against thrombin and trypsin are reported. We found that several compounds with neutral P(1) groups exhibit excellent selectivity over trypsin and good potency against thrombin. The SAR data of the P(1) groups obtained here can be used in preparing other thrombin inhibitors with better selectivity against trypsin.