Cellulose-acetate gel electrophoresis, a technique commonly used for the separation of human acid hydrolases, was applied to study heterogeneity in acid β-glucosidase (EC 3.2.1.45). With this technique, three forms of β-glucosidase were distinguisable in extracts of several tissues. the most anodic β-glucosidase activity (band 3) represents the broad-specificity β-glucosidase that is not deficient in Gaucher disease and is not inhibited by conduritol B-epoxide (CBD). The β-glucosidase activity near the origin (band 1) comigrated with puro monomeric glucocerebrosidase and was inhibited by CBE; this activity was deficient in Gaucher disease. A third β-glucosidase activity with an intermediate mobility (band 2) was also inhibited by CBE and deficient in Gaucher disease. Band 1 and band 2 β-glucosidase thus represent different forms of glucocerebrosidase. By adding phosphatikylserine and aphingolipid activator protein (SAP-2), monomeric glucocerebrosidase could be completely converted into a form that comigrated with band 2 β-glucosidase of tissue extracts. The addition of phosphatidylserine only also resulted in a changed mobility of the monomeric enzyme, but the migration in this case differed from that of band 2 β-glucosidase of tissue extracts. The electrophoretic profile of β-glucosidase activity of tissue extracts changed upon ethanol/chloroform extraction: the two glucocerebrosidase forms were converted into a band with a mobility identical to that of band 1 β-glucosidase. Our findings indicate that the interaction of glucocerebrosidase with phospholipid and SAP-2 has major effects on mobility of the enzyme in the cellulose-acetate gel electrophoresis system. The findings with the cellulose-acetate gel electrophoretic system are discussed in relation to the heterogeneity in glucocerebrosidase observed with sucrose density gradient analysis, immunochemical methods and isolelectric focussing studies.