Pyruvate dehydrogenase mutants of Saccharomyces cerevisiae were isolated by disruption of the PDAl gene. To this end, the PDAl gene encoding the E1α subunit of the pyruvate dehydrogenase complex was replaced by the dominant Tn5ble marker. Disruption of the PDAl gene abolished production of the E1α subunit and pyruvate dehydrogenase activity. Two additional phenotypes were observed in the Pdh− mutants: (a) a reduced growth rate in glucose medium which was partially complemented by the amino acid leucine; (b) an increase in formation of petites which lack mitochondrial DNA [rho°], during growth on glucose. Both phenotypes were shown to be a result of inactivation of the PDAl gene. Explanations for these phenotypes are discussed.