The protein O-mannosyltransferases Pmt1p and Pmt2p are catalyzing the O-glycosylation of serine and threonine residues in the endoplasmic reticulum of yeast. Deletion of each of these proteins by disruption of the corresponding gene leads to a dramatic decrease of mannosyltransferase activity in vitro. With an anti-Pmt1p immunoaffinity column a complex of Pmt1p and a second protein was purified; this protein turned out to be Pmt2p. Overexpression of Pmt1p or Pmt2p, respectively, does not increase mannosyltransferase activity in vitro. Overexpression of both mannosyltransferases together, however, raises in vitro activity threefold. These data indicate that Pmt1p and Pmt2p function as a complex catalyzing protein O-glycosylation in yeast.