Abstract: The cell wall of yeast contains proteins (CWPs) that are covalently bound to the glycan network. These CWPs mediate cell-cell interactions and may be involved in cell wall biosynthesis. Using tandem mass spectrometry, we have identified 19 covalently bound CWPs of Saccharomyces cerevisiae. Twelve of them are shown for the first time to be covalently incorporated into the cell wall. The identified proteins include 12 predicted glycosylphosphatidylinositol-modified CWPs, all four members of the Pir-protein family, and three additional proteins (Scw4p, Scw10p, and Tos1p) that are, like Pir-proteins, connected to the cell wall glycan network via an alkali-sensitive linkage. However, Scw4p, Scw10p, and Tos1p do not contain internal repeat sequences shown to be essential for Pir-protein incorporation and may represent a separate class of CWPs. Strikingly, seven of the identified proteins (Gas1p, Gas3p, Gas5p, Crh1p, Utr2p, Scw4p and Scw10p) are classified as glycoside hydrolases. Phenotypic analysis of deletion mutants, lacking the corresponding CWP-encoding genes, indicated that most of them have altered cell wall properties, which reinforces the importance of the identified proteins for proper cell wall formation. In particular, gas1 and ecm33 mutants were highly sensitive to Calcofluor white and high temperature, whereas gas1, scw4, and tos1 mutants were highly resistant to incubation with beta-1,3-glucanase. The CWP identification method developed here relies on directly generating tryptic peptides from isolated cell walls, and is independent of the nature of the covalent linkages between CWPs and cell wall glycans. Therefore, it will probably be equally effective in many other fungi.