The polymerase chain reaction was used to amplify a novel fucosyltransferase cDNA (FucT-VI) from A431 and from HL60 cells. The amplified cDNA has a high degree of sequence identity to FucT-V and to FucT-III, and a much lower level of similarity to FucT-IV. Transfection of the FucT-VI gene into mammalian cells confers alpha-1,3 fucosyltransferase activity to the cells, resulting in cell surface expression of Lewis x and sialyl-Lewis x carbohydrates. In contrast to FucT-IV activity, FucT-VI catalyzes the transfer of fucose from GDP-beta-fucose to alpha-2,3 sialylated substrates. The substrate specificity of the FucT-VI gene product suggests that FucT-VI may be an enzyme involved in the biosynthesis of the E-Selectin ligand, sialyl-Lewis x, in myeloid cells.