Fibroblast activation protein (FAP) is a transmembrane serine peptidase that belongs to the prolyl peptidase family. FAP has been implicated in cancer; however, its specific role remains elusive because inhibitors that distinguish FAP from other prolyl peptidases like dipeptidyl peptidase-4 (DPP-4) have not been developed. To identify peptide motifs for FAP-selective inhibitor design, we used P2-Pro1 and acetyl (Ac)-P2-Pro1 dipeptide substrate libraries, where P2 was varied and substrate hydrolysis occurs between Pro1 and a fluorescent leaving group. With the P2-Prol library, FAP preferred Ile, Pro, or Arg at the P2 residue; however, DPP-4 showed broad reactivity against this library, precluding selectivity. By contrast, with the Ac-P2-Pro1 library, FAP cleaved only Ac-Gly-Pro, whereas DPP-4 showed little reactivity with all substrates. FAP also cleaved formyl-, benzyloxycarbonyl-, biotinyl-, and peptidyl-Gly-Pro substrates, which DPP-4 cleaved poorly, suggesting an N-acyl-Gly-Pro motif for inhibitor design. Therefore, we synthesized and tested the compound Ac-Gly-prolineboronic acid, which inhibited FAP with a Ki of 23 ± 3 nM. This was ∼9- to ∼5400-fold lower than the Ki values for other prolyl peptidases, including DPP-4, DPP-7, DPP-8, DPP-9, prolyl oligopeptidase, and acylpeptide hydrolase. These results identify Ac-Gly-BoroPro as a FAP-selective inhibitor and suggest that N-acyl-Gly-Pro-based inhibitors will allow testing of FAP as a therapeutic target. [ABSTRACT FROM AUTHOR]