The precise roles of protein glycosylation in multicellular development are poorly understood. We have characterized the mntA gene from Dictyostelium discoideum which encodes the beta-1,4-mannosyltransferase enzyme that catalyzes the reaction: GDP-Man + dolichol-PP-GlcNAc2 --> dolichol-PP-GlcNAc2-Man + GDP. This gene has a central role in the synthesis of the lipid-linked oligosaccharide precursor which becomes the core of all asparagine-linked (N-linked) glycans. The mntA gene contains a single small intron and encodes a 493 aa protein with a predicted molecular size of 56 kDa. It is located 5' to the repE gene on chromosome IV and is transcribed in the opposite orientation to repE with which it shares a 585 bp of upstream intergenic region. The predicted mntA gene product shares 38% homology with the S. cerevisiae ALG1 gene product. The MntA protein has a region homologous to the putative dolichol-binding region in the yeast ALG1 protein, but it is located in a different part of the molecule. Northern analysis revealed that the expression of the mntA gene is regulated during multicellular development with two periods of mRNA accumulation. The mntA gene product has a classical endoplasmic reticulum retention motif, and is the first Dictyostelium gene encoding a protein that is active in this organelle. The identification of this gene will allow expanded studies of the role of N-linked glycans in multicellular development.