Among five different human alpha 1 --> 3 fucosyltransferases cloned, fucosyltransferases III (Fuc-TIII) and IV (Fuc-TIV) differ significantly from each other. Fuc-TIII transfers a fucose to both sialylated and nonsialylated N-acetyllactosamine, but Fuc-TIV apparently transfers a fucose only to neutral N-acetyllactosamine. In this study, Chinese hamster ovary (CHO) cells were stably transfected with Fuc-TIII or Fuc-TIV, and the resultant cell lines, CHO-FTIII and CHO-FTIV, were compared for the carbohydrate structures and for their binding to E-selectin or L-selectin. CHO-FTIII and CHO-FTIV cells were labeled metabolically with [3H]galactose, and glycopeptides obtained from these cells were fractionated by serial lectin affinity chromatography. The fractionated glycopeptides were then subjected to various combinations of exoglycosidase treatment or endo-beta-galactosidase digestion. The results obtained can be summarized as follows. CHO-FTIII cells express sialyl Lewisx, Lewisx, and VIM-2 structures, whereas CHO-FTIV cells express only an Lex structure with a small amount of VIM-2 structure. When CHO-FTIII and CHO-FTIV cells were tested for adhesion to E-selectin expressed by tumor necrosis factor-activated endothelial cells and to an E-selectin chimeric protein, only CHO-FTIII cells were found to adhere well to E-selectin. Moreover, both CHO-FTIII and CHO-FTIV cells failed to adhere to an L-selectin chimeric protein. These results clearly indicate that FT-III and FT-IV direct distinctly different fucosylated oligosaccharides. This difference in oligosaccharide structures results in an entirely different efficiency in adhesion to E-selectin. The results also demonstrate that expression of sialyl Lex itself is not sufficient for L-selectin binding.