Résumé : The Lewis blood group-specified N-acetylglucosaminide α1 →4 fucosyltransferase and an N-acetylglucosaminide α1 →3 fucosyltransferase have been co-purified over 500,000-fold from human milk by affinity chromatography on GDP-hexanolamine agarose. The purified enzyme preparation migrates as two major bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with apparent M(r) = 53,000 and 51,000. Analysis of the acceptor substrate specificity of the transferase(s) and structural characterization of the reaction products indicate that the enzyme(s) forms the Fucα1→4GlcNAc, Fucα1 →3GlcNAc, and Fucα1 →3Glc linkages with oligosaccharide acceptors containing the nonreducing terminal sequences Ga1β1 →3GlcNAc, Ga1β1 →4GlcNAc, and Ga1β1 →4Glc, respectively. The two fucosyltransferase activities are activated to the same extent by a variety of divalent metal ions, inactivated at identical rates by thermal denaturation or reaction with N-ethylmaleimide, and inhibited to the same extent by rabbit antiserum prepared against the purified fucosyltransferase(s). In addition, kinetic analysis of the initial rate data obtained using acceptors for one of the fucosyltransferase activities as an inhibitor of the second suggests that acceptors for both fucosyltransferase activities bind at a single active site.