Tripeptide aldehydes such as Boc-D-Phe-Pro-Arg-H (51) exhibit potent direct inhibition of thrombin. This distinction offers important insight for the design of more potent and selective serine protease inhibitors which may be useful pharmacological tools and hold promise for development of clinically useful agents. The structure-activity relationships (SAR) on a series of anticoagulant peptides with high selectivity for the enzyme thrombin are discussed. The SAR is centered on a series of di- and tripeptide arginine aldehydes based on the structure of 51. The structural and conformational role of the amino acid residue in position 1 was investigated by substitution with conformationally restricted aromatic amino acids, aromatic acids, and a dipeptide isostere containing the psi[CH2N] amide bond replacement. Many of these peptides demonstrate potent antithrombotic activity along with selectivity toward thrombin, determined by comparison of in vitro inhibitory effects on trypsin, plasmin, factor Xa, and tissue plasminogen activator. Compound 5f, D-1-Tiq-Pro-Arg-H.sulfate is highly active and the most selective tripeptide aldehyde inhibitor of thrombin reported to date.