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The turnover of newly synthesized cytochromes P-450scc and P-45011 beta, and adrenodoxin was investigated in bovine adrenocortical cells in primary monolayer cultures. Cells were pulse-radiolabeled with [35S]methionine, and specific newly synthesized enzymes were immunoisolated at various times following labeling and quantitated. Adrenocorticotropin (ACTH) treatment did not alter the average turnover rate of total cellular proteins or that of total mitochondrial proteins. The half-life of total cellular proteins of control and ACTH-treated cells was determined to be 20.5 and 23 h, respectively. The half-life of mitochondrial proteins of control and ACTH-treated cells was determined to be 42.5 and 44 h, respectively. The turnover rate of newly synthesized cytochrome P-450scc was approximately the same as total mitochondrial protein (t1/2 = 38 h), and was unchanged by ACTH treatment (t1/2 = 42 h). ACTH treatment did not greatly alter the turnover rate of adrenodoxin. The half-life of adrenodoxin from control and ACTH-treated cells was determined to be 20 and 17 h, respectively. However, ACTH treatment appeared to increase the half-life of cytochrome P-45011 beta from 16 h in control cells to 24 h in treated cells. The differential rate of turnover of mitochondrial proteins studied here supports the contention that mitochondria are subject to heterogeneous degradation. It appears that chronic treatment of bovine adrenocortical cells in culture with ACTH leads to increased steroidogenic capacity, primarily as a result of increased synthesis of steroidogenic enzymes, although, as shown for cytochrome P-45011 beta, ACTH action might also increase steroidogenic capacity by increasing the half-life of this steroid hydroxylase.