To investigate the synthesis of alpha2-fucosylated epitopes in the bovine species, we have characterized cDNAs from various tissues. We found three distinct alpha2-fucosyltransferase genes, named bovine fut1, fut2, and sec1 which are homologous to human FUT1, FUT2, and Sec1 genes, respectively. Their open reading frames (ORF) encode polypeptides of 360 (bovine H), 344 (bovine Se), and 368 (bovine Sec1) amino acids, respectively. These enzymes transfer fucose in alpha1,2 linkage to ganglioside GM(1)and galacto- N -biose, but not to the phenyl-beta-D-galactoside, type 1 or type 2 acceptors, suggesting that their substrate specificity is different and more restricted than the other cloned mammalian alpha2-fucosyltransferases. Southern blot analyses detected four related alpha2-fucosyltransferase sequences in the bovine genome while only three have been described in other species. The supernumerary entity seems to be related to the alpha2-fucosyltransferase activity which can also use type 1 and phenyl-beta-D-galactoside substrate acceptors. It was exclusively found in bovine intestinal tract. Our results show that, at least in one mammalian species, four alpha2-fucosyltransferases are present, three adding a fucose on alpha1,2 linkage on type 3/4 acceptor (Galbeta1-3GalNAc) and another able to transfer also fucose on phenyl-beta-D-galactoside and type 1 (Galbeta1-3GlcNAc) acceptors. The phylogenetic tree of the enzymes homologous to those encoded by the bovine fut1, fut2, and sec1 genes revealed two main families, one containing all the H-like proteins and the second containing all the Se-like and Sec1-like proteins. The Sec1-like family had a higher evolutionary rate than the Se-like family.