The authors show that the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of Saccharomyces cerevisiae, previously thought to be restricted to the cell interior, is also present in the cell wall. GAPDH activity, proportional to cell number and time of incubation, was detected in intact wild-type yeast cells. Intact cells of yeast strains containing insertion mutations in each of the three structural TDH genes (tdh1, tdh2 and tdh3) and double mutants (tdh1 tdh2 and tdh1 tdh3) also displayed a cell-wall-associated GAPDH activity, in the range of parental wild-type cells, although with significant differences among strains. A cell wall location of GAPDH was further confirmed in wild-type and tdh mutants by indirect immunofluorescence and flow cytometry analysis with a polyclonal antibody against S. cerevisiae GAPDH. By immunoelectron microscopy, the GAPDH protein was detected at the outer surface of the cell wall of wild-type cells, as well as in the cytoplasm. Western immunoblot analysis of cell wall extracts and cytosol showed that Tdh2 and Tdh3 polypeptides are present in the cell wall, as well as in the cytosol, of exponentially growing cells. Tdh1 is only detected in stationary-phase cells, again in both cytosol and cell wall extracts. The results incorporate the GAPDH of S. cerevisiae, encoded by TDH1-3, into the newly emerging family of multifunctional cell-wall-associated GAPDHs which retain their catalytic activity.