Cystathionine β-lyase (CBL) catalyzes the hydrolysis of L-cystathionine (L-Cth) to produce L-homocysteine, pyruvate, and ammonia. A series of site-directed variants of Escherichia coli CBL (eCBL) was constructed to investigate the roles of the hydroxyl moieties of active-site residues Y56, Y111, Y238, Y338, and S339 as determinants of specificity. The effect of these conservative substitutions on the k(cat)/K(m)(L-Cth) for the α,β-elimination of L-Cth ranges from a change of only 1.1-fold for Y338F to a reduction of 3 orders of magnitude for the alanine replacement variant of S339. A novel role for residue S339 as a determinant of reaction specificity, via tethering of the catalytic base, K210, is demonstrated. Comparison of the kinetic parameters for L-Cth hydrolysis with those for the inhibition of eCBL by aminoethoxyvinylglycine (AVG) indicates that Y238 interacts with the distal carboxylate group of the substrate. The 22 and 50-fold increases in the K(m)(L-Cth) and K(i)(AVG) resulting from replacement of Y56 with phenylalanine suggest that this residue may interact with the distal amino group of these compounds, although an indirect role in binding is more likely. The near-native k(cat)/K(m)(L-Cth) and pH profile of the eCBL-Y111F variant demonstrate that residue Y111 does not play a role in proton transfer. The understanding of the eCBL active site and of the determinants of substrate and reaction specificity resulting from this work will facilitate the design of inhibitors, as antibacterial therapeutics, and the engineering of enzymes dependent on the catalytically versatile pyridoxal 5'-phosphate cofactor to modify reaction specificity.