The expression of the Lewis X (Lex) carbohydrate structure in brain is developmentally regulated and is thought to play a role in cell-cell interaction during neuronal development. Mice possess three functional alpha1,3-fucosyltransferase genes: Fut4, Fut7, and Fut9. Fut7 is known to have no activity to synthesize Lex. In the present study, the relative activities of Fut4 and Fut9 for Lex synthesis were determined using recombinant enzymes. Fut9 exhibited very strong activity for oligosaccharide acceptors and glycolipid acceptors, that is, more than 10- and 100-fold, respectively, than that of Fut4. Furthermore, both cerebrum and cerebellum at various stages of development (E17, P0, P7, P30, P100) expressed 15-100 times more Fut9 transcript than Fut4 transcript. Neurons and astrocytes in primary culture also expressed 10-15 times more Fut9 than Fut4 transcript. Moreover, alpha1,3-Fut activity toward a polylactosamine chain in homogenates of brain tissues and primary cultured cells showed a pattern typical of Fut9, not Fut4. The developmental profile of activity for the synthesis of Lex was well correlated with that of Fut9 transcript. Immunohistochemistry with anti-Fut9 monoclonal antibody revealed the distribution of the Lex structure. These results showed that Fut9 is the most responsible enzyme for the synthesis of Lex in brain.