Three distinct forms of beta-glucosidase were separated by Bio-Gel P-150 gel filtration from the 105,000 x g supernatant of liver, placenta and fibroblasts extracted in the presence of Cutscum and sodium taurocholate. Glucosylceramide beta-glucosidase was mostly particulate (more than 80%), had an apparent molecular weight of about 67,000 and exhibited a pH optimum of 5.9 with the natural substrate, glucosylceramide, and a pH optimum of 4.2 with the artificial substrate, 4-methylumbelliferyl beta-D-glucoside. This enzyme form was deficient in tissue specimens from patients with Gaucher disease. A soluble form with neutral pH optimum (6.2) was found to occur in high levels in liver and placenta but was relatively low in skin fibroblasts. This beta-glucosidase form had an apparent molecular weight of about 42,000 and was normal in the Gaucher tissues examined. Another neutral beta-glucosidase was found in the membranous fraction, it had a molecular weight greater than 150,000, cleaved 4-methylumbelliferyl beta-D-glucoside but not glucosylceramide, with an optimum pH of about 5.7. The membranous acidic and neutral forms could not be precipitated by antibodies to the soluble neutral beta-glucosidase. The three enzyme forms differed from one another by their heat inactivation profiles, the soluble neutral form was most labile, the membranous neutral form was most stable, and the acidic form with glucosylceramide beta-glucosidase activity had intermediate thermostability.