The covalent intermediate formed during catalysis by the lac Z beta-galactosidase from Escherichia coli can be trapped by reaction of the enzyme with 2',4'-dinitrophenyl 2-deoxy-2-fluoro-beta-D-galactopyranoside, thereby inactivating the enzyme. Kinetic parameters for this inactivation process with the holo- and apo-enzymes have been determined. The intermediate so formed turns over only very slowly (t1/2 = 11.5 h) resulting in reactivation of the enzyme. The nucleophilic amino acid involved has been identified as Glu-537 by using a tritium-labeled inactivator to label the enzyme, then cleaving the labeled protein into peptides and purifying and sequencing the labeled peptide. This residue is conserved in five homologous beta-galactosidases and is different from that (Glu-461) proposed to be the nucleophile (Herrchen, M., and Legler, G. (1984) Eur. J. Biochem. 138, 527-531) on the basis of affinity labeling studies with conduritol C cis-epoxide. A role for glutamic acid residue 461 as the acid/base catalyst is proposed and justified.