The CD15 carbohydrate epitope is expressed in mature human neutrophils, monocytes, and promyelocytes. We aimed to determine the α 1,3-fucosyltransferase responsible for the expression of CD15 in each subpopulation of leukocytes. Three α1,3-fucosyltransferases, FUT4, FUT7, and FUT9, are expressed in human leukocytes. We demonstrated that FUT9 exhibits 20-fold stronger activity for CD15 synthesis than FUT4, whereas FUT4 exhibits 4.5-fold stronger activity for CDw65 synthesis than FUT9. By competitive reverse transcriptase-polymerase chain reaction, FUT9 was found to be strongly expressed in mature granulocytes and peripheral blood mononuclear cell, but not in monocytes. CD34+ and CD15+ cells in cord blood and myeloid cell lines (HL-60 and U937) did not express FUT9 at all. FUT4 transcripts were ubiquitously expressed in all blood cells and all cultured cell lines, with HL-60 and U937 cells in particular expressing a number of FUT4 transcripts. Transfection of the FUT9 gene into Jurkat and U937 cells demonstrated that FUT9 has the potential to express CD15 in myeloid and lymphoid cells. These findings suggest that the expression of CD15 in mature granulocytes is directed by FUT9, whereas it is determined in promyelocytes and monocytes by FUT4. Measurement of CD15 synthesizing activity in cell homogenates of each cell population using the polylactosamine acceptor further supported these conclusions.