beta-Glucosidase from Flavobacterium meningosepticum (Fbgl) catalyses the hydrolysis of beta-1,4-glucosidic bonds via a two-step double-displacement mechanism in which two amino acid residues act as nucleophile and acid/base catalyst. Definitive identification of these two residues is provided by the two active-site-directed inactivators, 2',4'-dinitrophenyl-2-deoxy-2-fluoro-beta-D-glucoside (2FDNPG) and N-bromoacetyl-beta-D-glucosylamine (NBGN), which stoichiometrically label the nucleophile and the acid/base catalyst of Fbgl, respectively. Pseudo-first-order inactivation rate constants (k(i)) of 0.25+/-0.01 and 0.05+/-0.01 min(-1) and dissociation constants (K-i) of 90+/-15 and 4.4+/-0.2 mM are determined for 2FDNPG and NBGN, respectively. Proteolytic digestion of the labelled proteins, followed by peptide mapping and tandem MS analysis identify Asp-247 and Glu-473 as the catalytic nucleophile and acid/base residues, respectively, of Fbgl. This study confirms that the catalytic nucleophile of family 3 glycohydrolase is conserved across sub-families. However, different sub-families may have unique general acid/base catalysts.