This investigation of sweel almond (beta)-glucosidase using various effectors and substrates provided information on the active site geometry and the mechanism of action. A variety of phenols inhibit the enzyme. The pH dependence of the K(,i)'s of phenol and of pentafluorophenol showed that the phenoxide form cannot bind to the enzyme, indicating the presence of an anionic site. With a series of phenols, (beta) = -.25, indicating that the phenols hydrogen bond to this anion. Although p-nitroanisole does not bind at this phenol site, which is the aglycone portion of the substrate binding site, it does bind to the enzyme in at least two sites. Multiple inhibition studies with methanol, dithiothreitol and p-nitroanisole showed that p-nitroanisole binds near the glucosidic bond and prevents dithiothreitol but not methanol binding Multiple inhibition studies also showed that pentafluorophenol cannot bind simultaneously with any of a variety of glucose analogs. This indicates that stringent steric requirements would prevent the hydrolysis products, glucose and phenoxide, from binding simultaneously to the enzyme. The solvent isotope effect of 1 for all substrates tested indicates that there is no protonation involved in or before the rate limiting step, and that the phenoxide is the immediate product of the reaction The hydrolyses of the p-nitrophenyl oxygen and thiol glucosides were shown to be catalyzed by the same enzyme by a number of criteria. k(,cat)/K(,m) for both substrates shows a pH dependence on two enzyme groups, postulated to be carboxyls, with pK(,a) values of approximately 4.5 and 6.7, where it appears that the pK(,a) 4.5 groups must be unprotonated and the pK(,a) 6.7 groups protonated for maximum activity. These reactions also involve basically the same mechanism, with cleavage of the glucoside at the glucose carbon-aglycone bond and with common glucosyl-enzyme intermediates. The activation seen with the thiol substrate is postulated to result from effects on binding of the substrate and from a decrease in the formation of less reactive intermediates