The methanolysis of benzylpenicillin is catalyzed by Enterobacter cloacae P99 class C β-lactamase and the pH dependence of kcat indicates that the catalytic groups involved of pKa ca. 5 and 10 are the same as those for hydrolysis. The kinetic solvent isotope effect (KSIE) is 1.42 for both the hydrolysis and methanolysis of benzylpenicillin. However, there is an inverse KSIE on of 0.83 ± 0.05 for the hydrolysis of benzylpenicillin and cephaloridine. There is also an abnormally high shift in the low pKa on going from H2O to D2O of 0.85 ± 0.15 from the pH dependence of both kcat and kcat/Km for both methanolysis and hydrolysis. The D2O shift on the high pKa of ca. 10 is the normal value of ca. 0.4. These results are consistent with a strongly hydrogen bonded system acting as the general base catalyst and low fractionation factors for the hydrogens involved. It is probable that this represents the tyrosine phenoxide ion hydrogen bonded to two lysine ammonium ions.