The Saccharomyces cerevisiae MNT1 gene encodes a Golgi mannosyltransferase. Gene disruption of the MNT1 locus leads to a greater than 90% reduction of specific alpha-1,2-mannosyltransferase activity with alpha-methylmannoside as acceptor. Null mutants of MNT1 are viable, have no apparent growth defect, and are blocked in the elongation of protein O-linked mannobiose. Structural analysis of the N-linked outer chain isolated from an mnn1 mnn10 mnt1 strain revealed no alteration in carbohydrate structure compared to the parental mnn1 mnn10 strain. The MNT1 gene is identical to KRE2, and mutations in the gene render cells resistant to the killer toxin K1 of S. cerevisiae, which suggests a role for O-mannosylated proteins in the resistance mechanism. In addition, MNT1 is part of a multigene family whose members are presumed to be yeast Golgi mannosyltransferases.