Pyruvate dehydrogenase complex, for the first time, was highly purified from commercial baker's yeast (saccharomyces cerevisiae). Proteolytic degradation was prevented by the inclusion of the protease inhibitors pepstatin A, leupeptin, and phenylmethanesulfonyl fluoride during the enzyme purification. The yield from 1 kg of pressed yeast was about 15--20 mg enzyme with a specific activity of 17--30 U/mg. Most of the kinetic and regulatory properties of the yeast enzyme were found similar to those of the mammalian mitochondrial pyruvate dehydrogenase complexes except that Km for pyruvate, when assayed at the pH optimum, was much higher than in the mammalian complexes and resembled the values reported for the complexes of gram-negative bacteria. Furthermore, neither in yeast homogenates nor in the isolated yeast pyruvate dehydrogenase complex, was any evidence found for regulation by interconversion (phosphorylation-dephosphorylation) as occurs in mammals, plants, and Neurospora crassa.