Macrocyclic pentapeptide analogues (5-9) of the sponge natural product cyclotheonamide A (CtA, 3) were prepared by means of our convergent [3 + 2] synthetic protocol, in which a late-stage primary amine group is available for substitution (Maryanoff et al. Proc. Natl Acad. Sci. U.S.A. 1993, 90, 8048). These analogues, as well as CtA and cyclotheonamide B (CtB, 4), were examined for their ability to inhibit the serine protease alpha-thrombin, in comparison with suitable reference standards. We characterized Michaelis-Menten and slow-binding kinetics for the cyclotheonamide derivatives. An attempt was made to utilize the unoccupied hydrophobic S3 subsite of thrombin via analogues 5 and 6. Also, removal of the hydroxyphenyl group, which is thought to be involved in an aromatic stacking interaction with Trp60D of thrombin, was explored via analogue 9. The importance of the alpha-keto and olefin groups was examined via 7 and 8, respectively. The relationship of structure and function with the analogues proved to be less predictable than anticipated.