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Y-1 adrenal tumor cells were incubated with aminoglutethimide with and without ACTH. Greater production of pregnenolone from endogenous cholesterol was observed (after washing to remove aminoglutethimide) in mitochondria from cells incubated with aminoglutethimide and ACTH than in those from cells incubated with aminoglutethimide alone. This response was inhibited by cycloheximide and puromycin but not by chloramphenicol or actinomycin D. ACTH increased the incorporation of [ 3 H]tyrosine into protein associated with mitochondria but not into total cell protein or protein of postmitochondrial supernatant. This response did not require aminoglutethemide block and was inhibited by cycloheximide and puromycin but not by chloramphenicol or actinomycin D. Dibutyryl cyclic AMP produced both of these responses (increased production of pregnenolone and synthesis of protein associated with mitochondria). The concentration of cycloheximide required to cause 50% inhibition of the responses to ACTH and dibutyryl cyclic AMP was approximately the same for steroidogenesis by whole cells, for production of pregnenolone by isolated mitochondria, for incorporation of [ 3 H]tyrosine into Y-1 cell protein and for the increase in synthesis of protein associated with mitochondria produced by ACTH (0.08-0.2 μg/ml). Disc gel electrophoresis revealed that the increased incorporation of [ 3 H]tyrosine involved two proteins corresponding to molecular weight of approximately 27,000 and 13,000, respectively. These observations suggest that ACTH promotes synthesis of protein(s) by cytoplasmic ribosomes on stable messenger RNA, that the protein(s) becomes associated with mitochondria and that the protein(s) includes one or more which are associated with the increase in production of pregnenolone produced in mitochondria by the addition of ACTH to adrenal cells.