In Saccharomyces cerevisiae, the structural genes PDC1, PDC5 and PDC6 each encode an active pyruvate decarboxylase. Replacement mutations in these genes were introduced in a homothallic wild‐type strain, using the dominant marker genes APT1 and Tn5ble. A pyruvate‐decarboxylase‐negative (Pdc−) mutant lacking all three PDC genes exhibited a three‐fold lower growth rate in complex medium with glucose than the isogenic wild‐type strain. Growth in batch cultures on complex and defined media with ethanol was not impaired in Pdc− strains. Furthermore, in ethanol‐limited chemostat cultures, the biomass yield of Pdc− and wild‐type S. cerevisiae were identical. However, Pdc− S. cerevisiae was unable to grow in batch cultures on a defined mineral medium with glucose as the sole carbon source. When aerobic, ethanol‐limited chemostat cultures (D = 0·10 h−1) were switched to a feed containing glucose as the sole carbon source, growth ceased after approximately 4 h and, consequently, the cultures washed out. The mutant was, however, able to grow in chemostat cultures on mixtures of glucose and small amounts of ethanol or acetate (5% on a carbon basis). No growth was observed when such cultures were used to inoculate batch cultures on glucose. Furthermore, when the mixed‐substrate cultures were switched to a feed containing glucose as the sole carbon source, wash‐out occurred. It is concluded that the mitochondrial pyruvate dehydrogenase complex cannot function as the sole source of acetyl‐CoA during growth of S. cerevisiae on glucose, neither in batch cultures nor in glucose‐limited chemostat cultures.