Cell wall proteins of Saccharomyces cerevisiae are anchored by means of a beta-1, 6-glucan-containing side-chain. It is not known whether this chain is linked to the protein part (e.g. through carbohydrate side-chains) or to the glycosylphosphatidylinositol (GPI) moiety of cell wall proteins. An IgA protease recognition site was introduced in Cwp2p, a beta-1, 6-glucosylated cell wall protein, immediately N-terminal from the omega amino acid (the attachment site of the GPI moiety). Proteolytic cleavage of this site revealed that the beta-1, 6-glucan epitope was not linked to the protein part. We conclude that neither N-or O-glycosylation is involved in beta-glucosylation of cell wall proteins. This confirms that the glycan core of the GPI moiety is the probable beta-1, 6-glucan attachment site.