A sulphated form of sialyl-Lewisx, NeuAcα2-3Galβ1-4(Fucα1-3)GlcNAc6OSO3β1-3Gal, was synthesized enzymatically from a precursor disaccharide, GlcNAc6OSO3β1-3Gal, using sequential steps involving β1,4-galactosyltransferase, α2,3-trans-sialidase and recombinant α1,3-fucosyltransferase, respectively. Successful enzymatic fucosylation at the 3 position of the GlcNAc6OSO3 residue demonstrated that fucosyltransferases are capable of generating, in situ, sulphated sialyl Lewisx structures containing sulphate at the 6 position of GlcNAc. The sulphated sialyl-Lewisx pentasaccharide produced by this procedure inhibited binding of a soluble form of L-selectin to 35SO4-labelled peripheral addressin with an IC50 of 0.8 mM, whereas sialyl-Lewisx tetrasaccharide was a weaker inhibitor, displaying an IC50 of 3.2 mM. Hemmerich and Rosen (Biochemistry, 33, 4820–4829, 1994) recently reported the presence of Galβ1-4GlcNAcO6SO3 structures on murine peripheral addressin Sgp50, in addition to sialyl Lewisx structures sulphated at the 6-O-galactose position. Based on our data, we suggest that sialyl Lewisx sulphated at the 6-O-GlcNAc position may also exist on receptors and function as a ligand for L-selectin.