The sialyl-Lewis X (SLe(x)) determinant is important in leukocyte extravasation, metastasis and bacterial adhesion. The role of the protein, N-glycan and O-glycan core structures for the biosynthesis of SLe(x) in vivo by fucosyltransferases (FucTs) is not known. Immunoglobulin G (IgG) Fc fusion proteins of alpha(1)-acid glycoprotein (AGP), P-selectin glycoprotein ligand-1 (PSGL-1) or CD43 were used to probe the specificity of FucT-III-VII expressed alone in 293T and COS cells or together with O-glycan core enzymes in Chinese hamster ovary (CHO)-K1 cells. Western blotting with the monoclonal antibodies CSLEX and KM93 showed that FucT-III and V-VII produced SLe(x) on core 2 in CHO cells. Only FucT-V, -VI and, with low activity, -VII worked on core 3 on CD43/IgG, but no SLe(x) was detected with CSLEX on PSGL-1/IgG with core 3. KM93 stained SLe(x) on core 2, but was not reactive with SLe(x) on core 3. FucT-III, V-VII made SLe(x) on N-glycans of AGP/IgG in CHO, but not in COS and 293T cells, even though the same FucTs could make SLe(x) on CD43/IgG and PSGL-1/IgG in these cells. Our results define the specificities of FucT-III-VII in SLe(x) biosynthesis on O-glycans with different core structures and the fine specificity of the widely used anti-SLe(x) monoclonal antibody, KM93.