Summary 1. The nature and intracellular distribution of the respiratory enzyme system of Azotobacter vinelandii has been investigated. 2. Whole cells of this organism were found to contain high concentrations of ubiquinone (Q-8) but, in contrast to Azotobacter vinelandii strain O, no menaquinone (vitamin K2) was detected. 3. Cell-free extracts prepared by means of the French pressure cell were resolved by differential centrifugation into large particles, small particles and highspeed supernatant. The respiratory chain was almost completely located in the particulate material and comprised flavin, ubiquinone and cytochromes c4 + c5, b1, a1 and α2. Ubiquinone was present in approx. 6-fold molar excess over the individual cytochrome components. 4. The unsupplemented small particles catalysed the rapid oxidation of a variety of substrates including succinate, reduced nicotinamide—adenine dinucleotide, L -malate and DL -lactate. 5. The highly active L -malate and DL -lactate oxidases present in these particles were not linked to pyridine nucleotide, but a low-activity nicotinamide—adenine dinucleotide-linked malate dehydrogenase ( L -malate:NAD+ oxidoreductase, EC 1.1.1.37) was detected in the high-speed supernatant. 6. The oxidation of DL -β-hydroxybutyrate was nicotinamide—adenine dinucleotide-linked and required the presence of both a soluble dehydrogenase ( D -3-hydroxy-butyrate:NAD+ oxidoreductase, EC 1.1.1.30) and a particulate cytochrome system. 7. Antimycin A and 2-alkyl-4-hydroxyquinoline-N-oxide inhibited respiration only at very high concentrations. Inhibition of nicotinamide—adenine dinucleotide oxidase was appreciably higher than succinate oxidase.