A gene encoding an α-1,2-mannosyltransferase from Saccharomyces cerevisiae was cloned and sequenced. The α-1,2-mannosyltransferase which utilizes α-methylmanno-side as acceptor of mannose from GDP-mannose was purified. The enzyme activity was shown to correspond to a 41 kDa protein band on sodium dodecyl sulphate–poly-acrylamide gel electrophoresis. This protein band was digested in situ with trypsin and amino acid sequence information was obtained from four peptides. Degenerate oligonucleotide primers corresponding to the amino acid sequences were designed and used for polymerase chain reactions on yeast genomic DNA. A specific reaction product was used to screen a genomic library of S.cerevisiae. A fragment of ∼5.7 kb was isolated, of which a 2.9 kb fragment was sequenced. It contained a 1329 base pair open reading frame encoding the peptide sequences of the purified α-1,2-mannosyhransferase. The gene, designated MNT1, is located on the right arm of chromosome 4. It encodes a 442 amino acid polypeptide with a calculated mol. wt of 51.4 kDa. The corresponding mRNA has a length of ∼1.6 kb. Overexpression of the MNT1 gene increased this α-1,2-mannosyltransferase activity ∼2.5-fold. The protein was shown to be modified with N-linked carbohydrate chains and its sequence contains one N-glycosylation site. The enzyme contains a putative membrane-spanning domain near its N-terminus and its topology is thus similar to that of mammalian Golgi glycosyltransferases. This is the first report of the cloning and sequencing of a yeast Golgi mannosyltransferase.