The adrenal gland mitochondrial cytochrome P-450 reductase system (adrenal flavoprotein and adrenodoxin) is shown in this investigation to enhance steroid 21 hydroxylation in carefully prepared microsomal suspensions and acetone powder extracts. Examination of the reductase components separately reveals that the enhancement can be accounted for on the basis of the flavoprotein moiety alone.This implicates flavoprotein as an electron carrier in steroid 21 hydroxylation. While it seems likely that the mitochondrial and microsomal flavoproteins are identical, there remains a possibility that different, yet compatible, species may exist for the two organelles. The current studies indicate that V^/teroid 11/3 hydroxylation was early shown to occur in adrenal gland cortex mitochondria (Sweat, 1951) and to require TPNH (Sweat and Lipscomb, 1955; Grant and Brownie, 1955) and molecular oxygen (Sweat et al., , 1956 Hayano et al., 1955a,b) as participants in the reaction. Shortly thereafter, 21 hydroxylation was shown to be associated with elements of the adrenal gland cortex endoplasmic reticulum (Plager and Samuels, 1954) and to also require TPNH (Ryan and Engel, 1956) and molecular oxygen (Cooper et al., 1963;Nakano et al., 1968). Both 11/3-and 21-hydroxylation reactions have been classified in the category of mixedfunction oxidases (Mason, 1957). While it has been suggested that steroid hydroxylations may occur as a generalized process, Mason (1957) has pointed out that nonspecific reactions characteristic of free radicals are not apparent in the steroid