Two genes PMT3 and PMT4 were identified by polymerase chain reaction of genomic DNA using primers derived from regions of high homology between the products of three genes PMT1, PMT2 of Saccharomyces cerevisiae and part of a PMT1 related sequence of Kluyveromyces lactis. Pmt1p and Pmt2p are mannosyltransferases involved in the transfer of a mannosyl residue from dolichyl phosphate-D-mannose (Dol-P-Man) to seryl and threonyl residues in proteins. The products encoded by the PMT3 and PMT4 genes have almost identical hydropathy profiles in comparison to PMT1 and PMT2: a hydrophobic N- and C-terminal third each with multiple potential transmembrane helices and a central hydrophilic part. The predicted Pmt3p contains 753 amino acids, four potential N-glycosylation sites and it is significantly homologous to Pmt1p, Pmt2p and Pmt4p. Pmt4p contains 762 amino acids and two potential N-glycosylation sites. Northern blot analysis showed a single mRNA transcript of PMT3 and PMT4 of 2.8 kb. Thus PMT3 and PMT4 are two new members of the PMT gene family. The pmt4 null mutant the pmt3 pmt4 double null mutant, but not pmt3 null mutant, showed a significant shift of chitinase due to under glycosylation of the enzyme. The triple disruption pmt2 pmt3 pmt4 and the quadruple disruption result in a lethal phenotype.