UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1, 2-N-acetylglucosaminyltransferase I (GnT I) is a key enzyme in the synthesis of Asn-linked complex and hybrid glycans. Studies on mice with a null mutation in the GnT I gene have indicated that N-glycans play critical roles in mammalian morphogenesis. This paper presents studies on N-glycans during the development of the nematode Caenorhabditis elegans. We have cloned cDNAs for three predicted C. elegans genes homologous to mammalian GnT I (designated gly-12, gly-13, and gly-14). All three cDNAs encode proteins (467, 449, and 437 amino acids, respectively) with the domain structure typical of previously cloned Golgi-type glycosyltransferases. Expression in both insect cells and transgenic worms showed that gly-12 and gly-14, but not gly-13, encode active GnT I. All three genes were expressed throughout worm development (embryo, larval stages L1-L4, and adult worms). The gly-12 and gly-13 promoters were expressed from embryogenesis to adulthood in many tissues. The gly-14 promoter was expressed only in gut cells from L1 to adult developmental stages. Transgenic worms that overexpress any one of the three genes show no obvious phenotypic defects. The data indicate that C. elegans is a suitable model for further study of the role of complex N-glycans in development.