The cholera toxin B subunit (CTB), which consists of five identical polypeptides and adopts a pentameric structure, has been shown to bind to the GM1-gangliosides at the cellular surface. Recombinant CTB has attracted much attention due to its non-toxicity and potential as a strong immunogenic antigen and immuno adjuvant for both system and mucosal immune responses. In this study, CTB was expressed in Saccharomyces cerevisiae and the resulting recombinant CTB was extensively characterized. PCR and back-transformation into E. coli confirmed the presence of the CTB gene-containing plasmid in the transformants and northern analysis showed the presence of the CTB-specific transcript. Western blot analysis of the yeast-derived protein extract showed the presence of CTB with mobility similar to purified CTB from Vibrio cholerae suggesting that the expressed CTB assembled into the desired pentameric form. Quantitative ELISA revealed that the recombinant CTB comprised approximately $0.5{sim}1.3%$ of the total cell-free extract. In addition, $0.5{sim}2;mg$ of CTB protein per liter of cultured media was detected 1 day, at the earliest after cultivation. The GM1-ganglioside enzyme-linked immunosorbent assay (GM1-ELISA) confirmed that the yeast-derived CTB bound specifically to the GM1-ganglioside receptor, indicating that it retained its native function and pentameric form, which is required for binding to intestinal epithelial cell membrane glycolipid receptors. In addition to the development of a yeast-derived edible vaccine against cholera, this study regarding the expression and assembly of recombinant CTB into biologically active oligomers in recombinant S. cerevisiae enables the efficient production of a GRAS microorganism-based adjuvant, as well as the development of carriers for foreign vaccine molecules.