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The action of adrenocorticotropin (ACTH) to stimulate synthesis of steroid 21-hydroxylase was studied in bovine adrenocortical cells maintained in primary culture. Continuous treatment with ACTH (1 microM) caused an increased incorporation of [35S]methionine into cytochrome P-450C21 (21-hydroxylase cytochrome P-450); a maximum value (15-fold increase) was attained 24 h after initiation of ACTH treatment. Also, a 3-fold increase in cytochrome P-450C21 synthesis was observed in a cell-free translation system programmed by RNA isolated from cells that were exposed to ACTH for 24 h. The rate of synthesis of cytochrome P-450C21 declined after longer periods of treatment of the cells with ACTH. The increase in synthesis of cytochrome P-450C21 was associated with an increase (3-6 fold) in both total cytochrome P-450 content and in the type I absorbance change induced by 17 alpha-hydroxyprogesterone in microsomes prepared from ACTH-treated cells, as compared with that in microsomes from control cells. By contrast, ACTH did not act to increase steroid 21-hydroxylase activity in cultured intact cells, as determined by the rate of secretion of cortisol and 11-deoxycortisol, after addition of 17 alpha-hydroxyprogesterone to the medium. Similarly, there was no difference in steroid 21-hydroxylase activity in postmitochondrial supernatant fractions prepared from non-treated or ACTH-treated cells. Cytochrome P-450C21 was found to be synthesized as a form identical in molecular weight to the mature form. These results are indicative that ACTH acts to stimulate the synthesis of steroid 21-hydroxylase, yet is without a demonstrable effect on the activity of this enzyme which is high throughout the time period of the experiment.