The formation of N-arylacetohydroxamic acids from nitroso aromatic compounds in the presence of pyruvate was investigated using isolated spinach leaf cells. The activity was enhanced by the addition of TPP, MgSO(4), and pyruvate, requirements for pyruvate dehydrogenase complex (PDHC). Measurement of the kinetic parameters revealed that the K(m) values of nitroso aromatic compounds tested were identical and that electron-donating ring substituents decreased the catalytic efficiency. The activation energy of the formation of N-phenylacetohydroxamic acid was lower than that reported for porcine heart PDHC. With alpha-oxo acids tested, alpha-oxobutyrate served as a substrate to give the corresponding N-phenylpropionylhydroxamic acid. The activity of spinach leaf cells in N-phenylacetohydroxamic acid formation was found in both mitochondria and chloroplasts. The contribution of chloroplast PDHC to total activity in the formation of N-phenylacetohydroxamic acid was estimated to be 50% under the conditions used.