The effects of O-glycosylation inhibitors on the growth and differentiation of the human acute promyeloblastic leukemia cell line HL-60 were studied to examine whether the O-glycosylation is needed for HL-60 cells to differentiate into granulocyte-like cells or monocyte-macrophage-like cells. N-Acetyl-alpha-D-galactosaminides, inhibitors of mucin-type oligosaccharide synthesis, and N-acetyl-beta-D-galactosaminides did not affect either growth or differentiation. beta-D-Xylosides, the artificial initiators of glycosaminoglycan synthesis, also were tested. Only 4-methylumbelliferyl-beta-D-xyloside induced HL-60 cells, to differentiate, and they differentiated into granulocyte-like cells, assessed by reduction of nitroblue tetrazolium, Giemsa staining, and esterase double-staining. The aglycon portion of 4-methylum-belliferyl-beta-D-xyloside, 4-methylumbelliferone, caused the differentiation. Thus we could find a new drug that induces the differentiation of HL-60 cells.