Using Sephadex G-75 and DEAE-cellulose column chromatography, an 8270-Da glycopeptide (designated Fragment II) has been isolated from a cyanogen bromideformic acid digest of a heat-stable factor from Gaucher spleen which activates a lipid-depleted preparation of lysosomal glucocerebrosidase from human liver. Fragment II contains all of the activity present in the native heat-stable factor. Compared with the parent factor, Fragment II contains four fewer cysteine and methionine residues and one less of each of the following: aspartic acid, threonine, serine, valine, isoleucine, and leucine. Nearly all of the monosaccharides present in the parent heat-stable factor can be accounted for in Fragment II, including three glucosamine, three mannose, one sialic acid, and one fucose. By itself, Fragment II has little or no stimulatory activity; its major effect is to markedly increase the sensitivity of glucocerebrosidase to activation by phosphatidylserine. A mixture of 1 μg phosphatidylserine and 2 μg of the cyanogen bromide fragment activates the lipid-depleted preparation of glucocerebrosidase 50% more than 30 μg phosphatidylserine alone. Analysis of the Km and Vmax of glucocerebrosidase at various hydrogen ion concentrations revealed that the heat-stable factor and phosphatidylserine together dramatically increase the catalytic efficiency ( Vmax Km) of glucocerebrosidase while making apparent three ionizable groups that participate in the catalysis. Phosphatidylserine alone recruits two ionizable groups, but catalytic efficiency is lower than when heat-stable factor is also present. Heat-stable factor alone has no discernable effect on the ionization of functional groups on the enzyme or on catalytic efficiency. By sucrose density gradient ultracentrifugation, it was shown that preincubation of rat liver glucocerebrosidase with phosphatidylserine and heat-stable factor shifted the enzyme completely from a 56,600-Da form to a 188,100-Da form. The activity of the slower sedimenting form of glucocerebrosidase was totally dependent upon exogenous bile salt activator, whereas the faster sedimenting form exhibited the same activity in the presence or absence of sodium taurocholate. It appears that the heat-stable factor promotes the transfer of phosphatidylserine to glucocerebrosidase, which, in turn, results in an increase in both the catalytic efficiency and size of the enzyme.