We cloned three members of a GDP-fucose:beta-galactoside alpha1,2-fucosyltransferase (alpha1,2-fucosyltransferase) family, MFUT-I, -II, and -III, from a cDNA of murine small intestine, and determined their enzymatic properties after transfection of the genes into COS-7 cells, and their expression in murine tissues by Northern blotting. MFUT-I, -II, and -III exhibited sequence homology with the human H (78.4%), Se (79.0%), and Sec1 (74.9%) gene products, respectively. COS-7 cells transfected with MFUT-I and -II exhibited alpha1,2-fucosyltransferase activity and the best acceptor substrate for both gene products was GA1 to yield a fucosyl GA1 structure, but no activity was detected in COS-7 cells with MFUT-III. MFUT-II yielded a 3.5-kb mRNA transcript in several tissues, whereas MFUT-I and -III were predominantly expressed in epididymis and testis, respectively. The administration of microbes into germ-free mice resulted in a rapid increase of the MFUT-II gene (Se gene) for the synthesis of fucosyl GA1 in the intestine.