A steroid 11β-hydroxylation system was reconstituted from NADPH-adrenodoxin re-ductase, adrenodoxin, and a P-450 preparation which had been extracted and partially purified from mitochondria of bovine adrenal cortex. 1. Oxidation of NADPH by the reconstituted system was accelerated by addition of deoxycorticosterone, and the formation of corticosterone was detected both chromatographically and fluorometrically. The reductase, adrenodoxin, and the P-450 preparation were all indispensable for the hydroxylation reaction. 2. NADPH oxidation activity in the presence and absence of deoxycorticosterone was measured under various conditions. The results indicated that in the oxidation of NADPH in the absence of deoxycorticosterone so-called “leakage of electrons” is predominant when the concentration of reduced adrenodoxin was relatively low. Acceleration of the oxidation of NADPH by deoxycorticosterone increases with the concentration of reduced adrenodoxin. “Leakage of electrons” is maximal at pH values around 6.5, whereas acceleration of oxidation by deoxycorticosterone is maximal at about pH 7.5. 3. Of the steroids tested only those inducing a type I difference spectrum of P-450 accelerated the oxidation of NADPH by the reconstituted system. The specificity of the reconstituted system for steroids, as measured by their acceleratory effects, agreed essentially with that of adrenal cortex mitochondria. 4. Pregnenolone, which induced a type II difference spectrum, inhibited the “leakage of electrons, ” but did not affect the acceleration of NADPH oxidation by deoxycorticosterone. 5. These results are most easily explained by supposing that P-450 existed in two states in the preparation, one of which is responsible for the “leakage” and the other for the hydroxylation reaction. 6. Considerable activity for cleavage of the cholesterol side chain was detected in the reconstituted system using isotopically labelled cholesterol.