Botulinum neurotoxins (BoNTs A-G) are zinc metalloendoproteases that exhibit extraordinary specificities for proteins involved in neurotransmitter release. In view of the extreme toxicities of these molecules, their applications in human medicine, and potential for misuse, it is of considerable importance to elucidate the mechanisms underlying substrate recognition and to develop inhibitors, with the ultimate goal of obtaining anti-botulinum drugs. We synthesized peptides based on vesicle-associated membrane protein (VAMP) to investigate the substrate requirements of BoNT F, which cleaves VAMP between residues Q58 and K59. The minimum substrate was a peptide containing VAMP residues 32-65, which includes only one of the two VAMP structural motifs thought to be required for botulinum substrate recognition. BoNT F exhibited a strict requirement for residues D57 (P(2)), K59 (P(1)'), and L60 (P(2)'), but peptides containing substitutions for R56 (P(3)), Q58 (P(1)), and S61 (P(3)') were cleaved. Therefore, the P(2), P(1)', and P(2)' residues of VAMP are of paramount importance for BoNT F substrate recognition near the scissile bond. K(i) values of uncleavable analogues were similar to K(m) values of the substrate, suggesting that substrate discrimination occurs at the cleavage step, not at the initial binding step. We then synthesized inhibitors of BoNT F that incorporated d-cysteine in place of glutamine 58, exhibited K(i) values of 1-2 nM, and required binding groups on the N-terminal but not the C-terminal side of the zinc ligand. The latter characteristic distinguishes BoNT F from other zinc metalloendoproteases, including BoNTs A and B.