Pyruvate dehydrogenase was partially purified from Ehrlich ascites tumor cell mitochondria and its kinetic properties were determined. The apparent KM values for pyruvate, nicotinamide adenine dinucleotide, and coenzyme A (CoA) were 46 muM, 110 muM, and 36 muM, respectively. Reduced nicotinamide adenine dinucleotide and acetyl-CoA inhibited enzyme activity competitively to nicotinamide adenine dinucleotide (Ki = 22 muM) and CoA (Ki = 58 muM), respectively. Copurified alpha-ketoglutarate dehydrogenase displayed apparent KM values for alpha-ketoglutarate, nicotinamide adenine dinucleotide, and CoA of 1.25 mM, 67 muM, and 50 muM, respectively. Pyruvate dehydrogenase, but not alpha-ketoglutarate dehydrogenase, was inactivated specifically by adenosine triphosphate with concomitant phosphorylation, and it was reactivated at 10 mM Mg2+ by a protein fraction separated from the complex during purification. The rate of inactivation was decreased by pyruvate or pyrophosphate. The existence of active and inactive forms of pyruvate dehydrogenase in Ehrlich ascites tumor cells was demonstrated. Active form and total activity were determined to be 74.0 +/- 1.5 and 93.6 +/- 4.9 munits/g packed cells (mean +/- S.E., n = 25), respectively.