Open reading frames (ORFs) in the genome of Saccharomyces cerevisiae were screened for cell wall proteins and extracellular proteins, using an in silico sequence analysis combined with biochemical examination. The selection criteria used in the sequence analysis were the presence of a signal sequence for secretion and the absence of any targeting and retention signal to/in intracellular components. By using the PSORT II program, 163 ORFs/proteins were selected as potential extracellular proteins, including cell wall proteins. Of these, 51 ORFs/proteins of unknown localization and more than 120 amino acids in size were further studied on their cellular localization. A hemagglutinin (HA) epitope was inserted in the most C-terminus of each protein and the resulting HA-tagged protein was expressed under the authentic promoter in yeast cells. Out of the 51 constructs, 35 gave protein bands on Western blots. Examination of proteins in fractionated samples identified 11 extracellular proteins; six proteins that were weakly associated with the cell wall and five proteins that were relatively tightly associated with the cell wall.