Cell wall extracts from the double-mutant mnn1 mnn9 strain were used as the immunogen to obtain a monoclonal antibody (MAb), SAC A6, that recognizes a specific mannoprotein--which we have named Icwp--in the walls of cells of Saccharomyces cerevisiae. Icwp runs as a polydisperse band of over 180 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of Zymolyase extracts of cell walls, although an analysis of the secretory pattern of the mannoprotein shows that at the level of secretory vesicles, it behaves like a discrete band of 140 kDa. Immunofluorescence analysis with the MAb showed that Icwp lies at the inner layer of the cell wall, being accessible to the antibody only after the outer layer of mannoproteins is disturbed by treatment with tunicamycin. The screening of a lambda gt11 expression library enabled us to identify the open reading frame (ORF) coding for Icwp. ICWP (EMBL accession number YLR391w, frame +3) codes for 238 amino acids, of which over 40% are serine or threonine, and contains a putative N-glycosylation site and a putative glycosylphosphatidylinositol attachment signal. Both disruption and overexpression of the ORF caused increased sensitivities to calcofluor white and Congo red, while the disruption caused an increased sensitivity to Zymolyase digestion, suggesting for Icwp a structural role in association with glucan.