The Saccharomyces cerevisiae MNN4 gene, which is involved in mannosylphosphate transfer from GDP-mannose to N-linked oligosaccharide, has been cloned from a lambda phage containing a yeast chromosome XI DNA fragment. The MNN4 ORF encodes a protein of 1178 amino acids. The deduced amino acid sequence shows a topology of type II membrane proteins and has a unique repeated sequence of lysine and glutamic acid at the C-terminus. Disruption and overexpression of MNN4 led to a decrease and increase, respectively, of the mannosylphosphate content in cell wall mannans prepared from both mnn4 and wild type strains. A dramatic decrease of mannosylphosphate occurs in delta mnn4 mutans. The results from genetic and biochemical experiments combine to suggest that Mnn4p is required to mediate mannosylphosphate transfer in both the core and outer chain portions of N-linked oligosaccharides.