Abstract Four Saccharomyces cerevisiae mannan mutants have been analyzed to assess the effect of the mutations on the properties of the cells and to determine the complementation patterns. The mutants were defective in the formation of an α-1→3-mannosyltransferase (mnn1), two different α-1→2-mannosyltransferases (mnn2 and mnn3), and a mannosylphosphate transferase (mnn4). The latter mutant was obtained from S. cerevisiae 4484-24D, while the first three came from S. cerevisiae X2180-1A and X2180-1B in a previous study (Raschke, W. C., Kern, K. A., Antalis C., and Ballou, C. E. (1973) J. Biol. Chem. 248, 4660). These two parent strains possess easily distinguishable immunochemical determinants in their mannans, X2180 being characterized by a mannotetraose unit [αMan(1→3)αMan(1→2)αMan(1→2)Man] while 4484-24D has a mannosylphosphorylmannotriose unit [see PDF for equation] instead of the mannotetraose side chain. It was shown previously (Antalis, C., Fogel., S., and Ballou, C. E. (1973) J. Biol. Chem. 248, 4655) that the diploid cross between these two strains had on its surface mannan of the X2180 chemotype. We now find that the mnn1 mutants of X2180 make mannan of the 4484-24D chemotype, which indicates that the mannosylphosphate transferase gene is present in the X2180 strain but is not expressed on the cell surface in the presence of an active α-1→3-mannosyltransferase, either in the wild type X2180 haploid or in the heterozygous diploid of X2180 x 4484-24D. Thus, these two structural features of the mannan have an epistatic relationship, with the mannotetraose unit appearing to be dominant. The various mutant strains have been analyzed for their ability to bind alcian blue dye. Strain 4484-24D binds very strongly and X2180 very weakly, reflecting the relative surface accessibility of mannosylphosphate groups in their cell walls. On the other hand, the mutants lacking phosphate (4484-24D-1 because it had a defective mannosylphosphate transferase, and X2180-1A-5 because it made no side chains to which mannosylphosphate groups could be attached) did not bind the dye at all. Thus, in these strains of S. cerevisiae it is clear that the primary ability to bind this dye was directly related to the presence of mannosylphosphate groups on the cell surface or in the cell wall. The diploid formed from a cross of X2180-1A-5, and 4484-24D-1 had the properties of the X2180 wild type, while the X2180 mnn1 mutants bound the dye in a manner characteristic of the 4484-24D wild type, a result that is consistent with the increased amount or accessibility of the mannosylphosphate group in the mnn1 mutants. Tetrad analysis of the hybrid diploids obtained from crosses between the various mutants, and between the mutants and the wild type parent, gave preliminary evidence that mnn1, mnn2, and mnn4 mutants are not linked.