β-N-Acetylhexosaminidase of HL-60 cells was separated into two main forms, A and S, by chromatography on DEAE-cellulose. Analysis of developmental changes in the isoenzyme pattern was complicated by the fact that the specific activity of β-N-acetylhexosaminidase underwent a 6-fold change during the normal growth cycle. Two other lysosomal enzymes, β-galactosidase and α-mannosidase, behaved similarly. Induction of differentiation of HL-60 cells with dimethyl sulphoxide at a low cell density (3 x 105 cells/ml) had a greater effect on the abundance of α-subunits of β-N-acetylhexosaminidase, measured with 4-methylumbelliferyl-β-N-acetylglucosaminide 6-sulphate, than of β-subunits, measured with 4-methylumbelliferyl-β-N-acetylglucosamine, and resulted in an isoenzyme profile in which A and B were the major forms, with the levels of form S greatly decreased.