It has been well established that one factor influencing the rate of side-chain cleavage of cholesterol is the rate of delivery of the substrate, cholesterol, from depots in the cytoplasm to the inner mitochondrial membrane within the organelle. This process of intracellular transport consists of two steps: transport to the mitochondria and transport from outer to inner membrane. Transport to mitochondria requires the cytoskeleton and Ca(2+)-calmodulin but not newly synthesized protein. Transport within the mitochondria requires newly synthesized protein. Both steps are stimulated by the trophic hormones ACTH and LH, in their respective target organs. Since the steroidogenic responses to these hormones are inhibited by cycloheximide, it is proposed that the new protein(s) required for these responses are synthesized in the cytoplasm. Using an assay based on the production of pregnenolone by mitochondria from bovine adrenal cortex, a protein of mol. wt approximately 8200 (temporarily referred to as 8.2 K) was isolated. The protein was purified to homogeneity and found to possess the following properties: (i) 8.2 K accelerated the synthesis of pregnenolone by isolated mitochondria (ii) it promoted entry of cholesterol from the incubation medium into mitochondria (iii) 8.2 K accelerated the transport of cholesterol from outer to inner membrane (iv) it also promoted loading of P-450scc with cholesterol, when either mitoplasts of the inner mitochondrial membranes were incubated with 8.2 K in vitro. Moreover, (v) the synthesis of 8.2 K was increased by ACTH and (vi) the half-life of the protein was less than 2 min.(ABSTRACT TRUNCATED AT 250 WORDS)