We have recently shown that beside a general stimulation of most adrenal proteins, corticotropin induces a marked increase in a specific adrenal cytosolic protein, protein E. in intact and hypophysectomized rats. To further clarify the mechanisms by which corticotropin exerts its trophic action we have investigated the effects of cycloheximide, calcium and calcium chelator administration on intact and hypophysectomized animals. These substances were injected in rats with or without corticotropin, and slices of adrenal glands from control and treated animals were removed 5 h later, incubated with [<sup>14</sup>C]- or [<sup>3</sup>H]-leucine for 2 h, and cytosolic proteins analyzed by polyacrylamide gel electrophoresis using a dual labelling technique. When high doses of cycloheximide (higher than 500 µg) were injected in rats, incorporation of labelled leucine in adrenal slices of control and corticotropin-treated animals was inhibited. With 500 µg cycloheximide per rat, incorporation of labelled leucine in adrenal slices of control animals was normal, but the corticotropin stimulation of both protein E and total protein synthesis was inhibited. Lower doses of cycloheximide (100 µg per rat) completely inhibited the stimulatory effect of corticotropin on total protein synthesis but did not affect protein E synthesis, while after 50 µg per rat both stimulatory effects were preserved. The two higher doses of cycloheximide (500 and 100 µg per rat) could not completely block the steroidogenic effect of the hormone. The effects of calcium and calcium chelators were studied in 1-day hypophysectomized rats. Calcium alone or injected simultaneously with corticotropin has no effect. Calcium chelators injected simultaneously with corticotropin partially inhibited the stimulatory effects of corticotropin on steroidogenesis but totally inhibited stimulation of total protein synthesis, while the stimulation of protein E persisted. Our results show that after corticotropin, stimulation of protein E synthesis correlates better with steroidogenesis than with total protein synthesis.