Abstract: S. cerevisiae contains at least six genes (PMT1-6) for dolicholphosphate-D-mannose: protein-O-D-mannosyltransferases. The in vivo mannosylation of seven O-mannosylated yeast proteins has been analyzed in a number of pmt mutants. The results clearly indicate that the various protein O-mannosyltransferases have different specificities for protein substrates. Five of the proteins tested (chitinase, a-agglutinin, Kre9p, Bar1p, Pir2p/hsp 150) are mainly underglycosylated in pmt1 and pmt2 mutants, whereby qualitative differences exist among the various proteins. Two of the O-mannosylated proteins (Ggp1p and Kex2p) are not at all affected in pmt1 and pmt2 mutants but are clearly underglycosylated when PMT4 is mutated. Although the PMT4 gene product is shown to be responsible for O-mannosylating a Ser-rich region of Ggp1p in vivo, a penta-seryl-peptide is not an in vitro substrate for this transferase. A PMT3 mutation does affect O-mannosylation of chitinase only in the genetic background of a pmt1pmt2 double mutation, indicating that PMT1 and PMT2 can compensate for a deleted PMT3 gene.