Fatty acid amide hydrolase (FAAH) is a mammalian integral membrane enzyme that catabolizes several neuromodulatory fatty acid amides, including the endogenous cannabinoid anandamide and the sleep-inducing lipid oleamide. FAAH belongs to a large group of hydrolytic enzymes termed the amidase signature (AS) family that is defined by a conserved, linear AS sequence of approximately 130 amino acids. Members of the AS family display strikingly different substrate selectivities, yet the primary structural regions responsible for defining substrate recognition in these enzymes remain unknown. In this study, a series of unbranched p-nitroanilide (pNA) substrates ranging from 6 to 20 carbons in length was used to probe the acyl chain binding specificity of FAAH, revealing that this enzyme exhibits a strong preference for acyl chains 9 carbons in length or longer. A fluorophosphonate inhibitor of FAAH containing a photoactivatable benzophenone group was synthesized and used to locate a region of the enzyme implicated in substrate binding. Protease digestion and mass spectrometry analysis of FAAH-inhibitor conjugates identified the major site of cross-linking as residues 487-493. Site-directed mutagenesis revealed that a single residue in this region, I491, strongly influenced substrate specificity of FAAH. For example, an I491A mutant displayed a greatly reduced binding affinity for medium-chain pNA substrates (7-12 carbons) but maintained nearly wild-type binding and catalytic constants for longer chain substrates (14-20 carbons). Mutation of I491 to aromatic or more polar residues generated enzymes with relative hydrolytic efficiencies for medium- versus long-chain pNAs that varied up to 90-fold. Collectively, these studies indicate that I491 participates in hydrophobic binding interactions with medium-chain FAAH substrates. Additionally, the significant changes in substrate selectivity achieved by single amino acid changes suggest that FAAH possesses a rather malleable substrate binding domain and may serve, along with other AS enzymes, as a template for the engineering of amidases with novel and/or tailored specificities.