An electrophoretic system using cellulose acetate has been developed for the resolution of β-glucosidase isozymes (β-D-glucoside glucohydrolase, EC 220.127.116.11 and D-glucosyl-N-acylsphingosine glucohydrolase, EC 18.104.22.168) in human tissue homogenates. Electrophoresis of homogenates from normal and Type I Gaucher disease tissues revealed two fluorescent bands of β-glucosidase activity which corresponded to the acid and neutral isozymes separated by concanavalin A-Sepharose chromatography. The acid isozyme had only β-glucosidase activity, whereas the neutral isozyme also exhibited α-L-arabinosidase (α-L-arabino-furanoside arabinofuranohydrolase, EC 22.214.171.124), β-D-galactosidase (β-D galactoside galactohydrolase, , EC 126.96.36.199) and β-D-xylosidase (1,4-β-D-xylan xylohydrolase, EC 188.8.131.52) activities, using the appropriate 4-methylumbelliferyl glycoside. In homogenates of cultured skin fibroblasts, only the acid isozyme was observed which co-electrophoresed with the acidic activity in other tissue homogenates. The acidic activity in tissue and fibroblast homogenates from Type I Gaucher disease appeared to co-electrophorese with the acid isozyme in normal tissues, but had markedly reduced activity.