The N-linked galactomannans of Schizosaccharomyces pombe have pyruvylated Galbeta1,3-(PvGal) caps on a portion of the Galalpha1,2-residues in their outer chains (Gemmill, T. R., and Trimble, R. B. (1998) Glycobiology 8, 1087-1095). PvGal biosynthesis was investigated by ethyl methanesulfonate mutagenesis of S. pombe, followed by the isolation of cells devoid of negatively charged N-glycans by Q-Sepharose exclusion and failure to bind human serum amyloid P component, which acts as a lectin for terminal PvGal residues. Mutant glycans were characterized by lectin binding, saccharide composition, exoglycosidase sensitivity, and NMR spectroscopy. Restoration of the cell surface negative charge by complementation with an S. pombe genomic library led to the identification of five genes involved in PvGal biosynthesis, which we designated pvg1-pvg5. Pvg1p may be a pyruvyltransferase, since NMR of pvg1(-) mutant N-glycans revealed the absence of only the pyruvyl moiety. Pvg2p-Pvg5p are crucial for attachment of the Galbeta1,3-residue that becomes pyruvylated. Pvg3p is predicted to be a member of the beta1,3-galactosyltransferase family, and Pvg3p-green fluorescent protein labeling was consistent with Golgi localization. Predicted Pvg1p and Pvg3p functions imply that Galbeta1,3-is added to the galactomannans and is then pyruvylated in situ, rather than by an en bloc addition of PvGalbeta1,3-caps to the outer chain. Pvg4p-green fluorescent protein targeted to the nucleus, and its sequence contains a MADS-box DNA-binding and dimerization domain; however, it does not appear to solely control transcription of the other identified genes. Pvg2p and/or Pvg5p may contribute to an enzyme complex. Whereas a functional role for the PvGal epitope in S. pombe remains unclear, it is nonessential for either cell growth or mating under laboratory conditions.