The PMT2 gene from Saccharomyces cerevisiae was identified as FUN25, a transcribed open reading frame on the left arm of chromosome I (Ouellette, B. F. F., Clark, M. W. C., Keng, T., Storms, R. G., Zhong, W., Zeng, B., Fortin, N., Delaney, S., Barton, A., Kaback, D.B., and Bussey, H. (1993) Genome 36, 32-42). The product encoded by the PMT2 gene shows significant similarity with the dolichyl phosphate-D-mannose:protein O-D-mannosyltransferase, Pmt1p (EC 2.4.1.109), which is required for initiating the assembly of O-linked oligosaccharides in S. cerevisiae (Strahl-Bolsinger, S., Immervoll, T., Deutzmann, R., and Tanner, W. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 8164-8168). The PMT2 gene encodes a new protein O-D-mannosyltransferase. Yeast cells carrying a PMT2 disruption show a diminished in vitro and in vivo O-mannosylation activity and resemble mutants with a nonfunctional PMT1 gene. Strains bearing a pmt1 pmt2 double disruption show a severe growth defect but retain residual O-mannosylation activity indicating the presence of at least one more protein-O-mannosyltransferase.