Here we report the discovery of a unique fucosyltransferase (FT) in Caenorhabditis elegans. In studying the activities of FTs in extracts of adult C. elegans, we detected activity toward the unusual disaccharide acceptors Gal1-4Xyl-R and Gal1-6GlcNAc-R to generate products with the general structure Fuc␣1-2Gal1-R. We identified a gene encoding a unique ␣1,2FT (designated CE2FT-1), which contains an open reading frame encoding a predicted protein of 355 amino acids with the type 2 topology and domain structure typical of other glycosyltransferases. The predicted cDNA for CE2FT-1 has very low identity (5-10%) at the amino acid level to ␣1,2FT sequences in humans, rabbits, and mice. Recombinant CE2FT-1 expressed in human 293T cells has high ␣1,2FT activity toward the simple acceptor Gal-O-phenyl acceptor to generate Fuc␣1-2Gal-R, which in this respect resembles mammalian ␣1,2FTs. However, CE2FT-1 is otherwise completely different from known ␣1,2FTs in its acceptor specificity, since it is unable to fucosylate either Gal1-4Glc-R or free lactose and prefers the unusual acceptors Gal1-4Xyl-R and Gal1-6GlcNAc-R. Promoter analysis of the CE2FT-1 gene using green fluorescent protein reporter constructs demonstrates that CE2FT-1 is expressed in single cells of early stage embryos and exclusively in the 20 intestinal cells of L 1 -L 4 and adult worms. These and other results suggest that multiple fucosyltransferase genes in C. elegans may encode enzymes with unique activities, expression, and developmental roles.