The plot of k(cat)/K(m) against pH for the Bacillus cereus 569/H β-lactamase class B catalysed hydrolysis of benzylpenicillin and cephalosporin indicates that there are three catalytically important groups, two of pK(a) 5.6 ± 0.2 and one of pK(a) 9.5 ± 0.2. Below pH 5 there is an inverse second-order dependence of reactivity upon hydrogen ion concentration, indicative of the requirement of two basic residues for catalysis. These are assigned to zinc(II)-bound water and Asp-90, both with a pK(a) of 5.6 ± 0.2. A thiol, N-(2'-mercaptoethyl)-2-phenylacetamide, is an inhibitor of the class B enzyme with a K(i) of 70 μM. The pH-dependence of K(i) shows similar pH inflections to those observed in the catalysed hydrolysis of substrates. The pH-independence of K(i) between pH 6 and 9 indicates that the pK(a) of zinc(II)-bound water must be 5.6 and not the higher pK(a) of 9.5. The kinetic solvent isotope effect on k(cat)/K(m) is 1.3 ± 0.5 and that on k(cat) is 1.5. There is no effect on reactivity by either added zinc(II) or methanol. The possible mechanisms of action for the class B β-lactamase are discussed, and it is concluded that zinc(II) acts as a Lewis acid to stabilize the dianionic form of the tetrahedral intermediate and to provide a hydroxide-ion bound nucleophile, whereas the carboxylate anion of Asp-90 acts as a general base to form the dianion and also, presumably, as a general acid catalyst facilitating C-N bond fission.