Aron B. Fisher,* Antonio Scarpa, Kathryn F. LaNoue, David Bassett, and John R. Williamson abstract: Mitochondria were prepared from homogenates of rat lungs by modification of standard techniques. The mitochondrial preparation was characterized by its content of cytochromes, adenine and pyridine nucleotides, coenzyme A, and divalent cations. Rates of substrate oxidation were mea-sured polarographically. Succinate was oxidized at a rate of 53±9 nmol of Cb/min permg of protein while other substrates were oxidized less rapidly. The Ca2+ content of the mitochondrial preparation was of majorimportance in deter-mining rates of substrate oxidation. Addition of 100-200 µ Ca2+ during oxidation of NAD-linked substrates markedly decreased respiration rate; this effect was due to loss of free pyridine nucleotides from the mitochondria in the presence of Ca2+. On the other hand, very low levels of Ca2^(< 1 µ) stimulated oxidation of «-glycerophosphate. This effect was