In Sacchuromyces cerevisiue, the structural genes PDCI, PDCS 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 TnSble. A pyruvate-decarboxylase-negative (Pdc-) mutant lacking all three PDC genes exhibited a threefold 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. cerevisiue 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-') 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.