The molecular mechanism of serine protease catalysis was investigated using a reaction field theory combined with molecular orbital calculation. The active site of the protein was represented as a multidielectric system. The region of the so-called oxyanion hole Was characterized as a microscopic domain with a high dielectric constant and the other region as a relatively low polarizable medium. A representative substrate, methyl formate, is embeded in such a pseudo-protein matrix with its carbonyl bond directing toward the oxyanion hole mimic. The energy profile was obtained for alkaline hydrolysis of the substrate, using a computational methodology recently developed by us. It was found that the rate-limiting step of the reaction is the demethoxylation from the well-known tetrahedral intermediate, and its energy profile sensitively depends on the electrostatic nature of the surrounding protein matrix. In particular, the reaction field generated from the oxyanion hole contributes to electrostatically stabilizing the tetrahedral intermediate. However, the most important finding is that the transition state is destabilized by the presence of the oxyanion hole, leading to an increase in the activation energy. This result may be inconsistent with the conventional picture of serine protease hydrolysis.