NADPH-adrenodoxin reductase from steer adrenal cortex mitochrondria has been purified to homogeneity (on sodium dodecyl sulfate polyacrylamide gel electrophoresis) by chromatography on DEAE-cellulose, Sephadex, and hydroxylapatite. A molecular weight of 51,500 was determined from sodium dodecyl sulfate polyacrylamide gel electrophoresis, while sedimentation equilibrium ultracentrifugation gave a value of 49,500. All of the flavine present was identified as FAD; 1 mol/52,000 g of protein. The reductase contained 1.7% carbohydrate (using glucose as standard) by weight. Homogeneous adrenodoxin reductase exhibited a typical oxidized flavoprotein absorbance spectrum, with maxima at 270, 376, and 450 nm, and gave an absorbance ratio A450/A270 of 0.122-0.128 (depending on the preparation). Reduction of the flavoprotein with NADPH or dithionite gave progressive bleaching of the 450-nm peak. The reductase was absolutely required, in the presence of adrenodoxin, for electron transfer from NADPH to cytochrome c or to particulate cytochrome P450. Adrenodoxin refuctase is obligatory for reconstitution of 11beta-hydroxylation activity using deoxycorticosterone as substrate, and for the side-chain cleavage of 20alpha-hydroxycholesterol or cholesterol. The specific activity of the homogeneous preparation in cytochrome c reduction is at least 17,000 nmol min-1 mg of protein-1, corresponding to a turnover number of 850 min-1. No evidence for the existence of multiple forms or subunits was obtained.