The Saccharomyces cerevisiae och1 mutant shows a deficiency in the mannose outer chain elongation at the non-permissive temperature. We have cloned the OCH1 gene by complementation of temperature sensitive (ts) phenotype for growth. The integrant of OCH1 gene in the yeast chromosome can complement the ts phenotype and shows the same mapping position as that of the och1 mutation, indicating that the cloned gene is the true gene for mutation. The OCH1 gene disruptant is not lethal but ts for cell growth, and lacks mannose outer chains. The OCH1 gene sequence predicts a 55 kDa protein consisting of 480 amino acids. It contains four potential asparagine-linked (N-linked) glycosylation sites and a single transmembrane region near the N-terminus. In vitro translation/translocation analysis revealed that the large C-terminal region of the OCH1 protein is located at the lumenal side of microsomal membranes with some sugar modification, indicating a type II membrane topology. The OCH1 protein was detected in yeast membrane fractions as four forms of 58-66 kDa, which correspond to the size of a glycoprotein containing four N-linked sugar chains the length of which is almost the same or slightly larger than the inner core (Man8GlcNAc2) formed in the endoplasmic reticulum (ER). Finally, the OCH1 gene was found to encode a novel mannosyltransferase which specifically transfers [14C]mannose to the unique acceptor, the core-like oligosaccharide of cell wall mannan accumulated in the och1 disruptant.