The expressions of β1,3-N-acetylglucosamonyltransferase-2 and -8 (β3GnT-2, β3GnT-8),—the two main glycosyltransferases responsible for the synthesis of poly-N-acetyllactosamine (polyLacNAc) in glycans, and β3GnT-5 participating in the syntheses of sphingoglycolipids were studied in leukemia cell lines during differentiation using RT-PCR method. β3GnT-2 and β3GnT-8 distribute widely in six myeloid and monocytoid leukemia cell lines with different abundances, while β3GnT-4 was only present in NB4 cells. ATRA (all-trans retinoic acid) and dimethylsulfoxide (DMSO), which induce the differentiation of HL-60 and NB4 (two human acute myeloid leukemia cell lines) to myelocytic lineage, up-regulated these two enzymes with various degrees at 2 and 72 h of treatment. In HL-60 cells treated with ATRA, the increase of β3GnT-8 was more than β3GnT-2, while in NB4 cells treated with DMSO, the increase of β3GnT-2 was more than β3GnT-8. However, when HL-60 and NB4 were differentiated to monocytic lineage induced by phorbol 12-myristate 13-acetate the expressions of β3GnT-2 and β3GnT-8 showed no alterations or the increase of expressions was far less than those in myelocytic differentiation. By means of FITC-labeled tomato lectin affinity staining and flow-cytometry, it was found that the product of β3GnT-2 and -8, polyLacNAc was also increased on the cell surface of HL-60 and NB4 treated with ATRA or DMSO, but unchanged when treated with PMA. These results were in accordance with the up-regulation of the mRNAs of β3GnT-2 and -8. The expression of β3GnT-5, however, was not changed both in myelocytic and monocytic differentiations. The difference in the up-regulation of β3GnT-2 and -8, especially their products may become a useful index to discriminate the myelocytic and monocytic differentiation of leukemia cells.