Extracts of human spleen contain two immunologically distinguishable forms of glucocerebrosidase: form I is precipitable by polyclonal or monoclonal anti‐(placental glucocerebrosidase) antibodies, whereas form II is not [Aerts, J. M. F. G., Donker‐Koópman, W. E., Van der Vliet, M. F. K., Jonsson, L. M. V., Ginns, E. I., Murray, G. J., Barranger, J. A., Tager, J. M. & Schram, A. W. (1985) Eur. J. Biochem. 150, 565–574]. The proportion of form II glucocerebrosidase was high in extracts of spleen, liver and kidney and low in extracts of brain, placenta and fibroblasts. Furthermore, the proportion of form II enzyme was higher in a detergent‐free aqueous extract of spleen than in a Triton X‐100 extract of total spleen or splenic membranes. When form II glucocerebrosidase in a splenic extract was separated from form I enzyme by immunoaffinity chromatography and stored at 4°C, a gradual conversion to form I enzyme occurred. The conversion was almost immediate if 30% (v/v) ethylene glycol was present. In the denatured state both forms of glucocerebrosidase reacted with anti‐(placental glucocerebrosidase) antibodies. Form I glucocerebrosidase was stimulated by sodium taurocholate or sphingolipid‐activator protein 2 (SAP‐2), whereas form II enzyme exhibited maximal activity in the absence of the effectors. The pH activity profile of form II glucocerebrosidase was almost identical to that of form I enzyme in the presence of SAP‐2. In the native state, form I glucocerebrosidase had a molecular mass of 60 kDa whereas that of form II glucocerebrosidase was about 200 kDa. After gel‐permeation high‐pertormance liquid chromatography of splenic extracts, the fractions with form II glucocerebrosidase contained material cross‐reacting with both anti‐(placental glucocerebrosidase) and anti‐(SAP‐2) antibodies. Preincubation of form I glucocerebrosidase with SAP‐2 at pH 4.5 led to masking of the epitope on glucocerebrosidase reacting with monoclonal anti‐(placental glucocerebrosidase) antibody 2C7. Furthermore, preincubation of form I glucocerebrosidase with monoclonal antibody 2C7 prevented activation of the enzyme by SAP‐2. We propose that form I glucocerebrosidase is a monomeric form of the enzyme, whereas form II glucocerebrosidase is a high‐Mr complex of the enzyme in association with sphingolipid‐activator protein 2.