Using bioinformatics methods, we have previously identified Glu235 and Glu340 as the putative acid/base catalyst and nucleophile, respectively, in the active site of human glucocerebrosidase. Thus, we undertook site-directed mutagenesis studies to obtain experimental evidence supporting these predictions. Recombinant retroviruses were used to express wild-type and E235A and E340A mutant proteins in glucocerebrosidase-deficient murine cells. In contrast to wild-type enzyme, the mutants were found to be catalytically inactive. We also report the results of various studies (Western blotting, glycosylation analysis, subcellular fractionation, and confocal microscopy) indicating that the wild-type and mutant enzymes are identically processed and sorted to the lysosomes. Thus, enzymatic inactivity of the mutant proteins is not the result of incorrect folding/processing. These findings indicate that Glu235 plays a key role in the catalytic machinery of human glucocerebrosidase and may indeed be the acid/base catalyst. As concerns Glu340, the results both support our computer-based predictions and confirm, at the biological level, previous identification of Glu340 as the nucleophile by use of active site labeling techniques. Finally, our findings may help to better understand the molecular basis of Gaucher disease, the human lysosomal disease resulting from deficiency in glucocerebrosidase.