Site-directed mutants of human carbonic anhydrase III were used to examine the role of Thr-199 and its interaction with Phe-198 in the catalyzed hydration of CO2. Threonine-199 is a hydrogen bond acceptor for the zinc-bound water, and Phe-198 forms part of the hydrophobic side of the active-site cavity of carbonic anhydrase III. Catalytic activity for a total of five single and double mutants at residues 198 and 199 was determined by stopped-flow spectrophotometry and 18O exchange between CO2 and water measured by mass spectrometry. The replacement Thr-199-->Ala resulted in a 4-fold decrease in the kcat/Km for hydration of CO2. We tested the hypothesis that the 25-fold increase in the kcat/Km for hydration of CO2 accompanying the replacement Phe-198-->Leu in isozyme III is caused by changes in the interaction of Thr-199 with the zinc-bound water or the transition state for catalysis. Comparison of hydration of CO2 by the single and double mutants of isozyme III containing the replacements Thr-199-->Ala and Phe-198-->Leu was consistent with an interaction between these two sites.