We have investigated the action of a protein synthesis inhibitor on the ability of angiotensin II (AII) to stimulate steroid synthesis. Isolated bovine adrenal glomerulosa cells were incubated in the presence and absence of angiotensin and cycloheximide, and the effects of the inhibitor on six cellular processes were measured. Cycloheximide at 7 and 28 microM inhibited the ability of the hormone to stimulate aldosterone synthesis. These concentrations of cycloheximide blocked protein synthesis by 72 and 79% respectively. Cycloheximide did not block receptor binding of angiotensin, the effect of angiotensin on [32P]phosphate incorporation into phosphatidylinositol, nor the ability of the hormone to alter 45Ca2+ fluxes. Mitochondrial conversion of cholesterol to pregnenolone is thought to be the rate-determining step in corticosteroid synthesis. Mitochondria isolated from cells treated with angiotensin made pregnenolone at a higher rate than control mitochondria. Cycloheximide blocked this effect when it was present in the cell incubation medium with angiotensin. Cycloheximide added directly to mitochondria had no effect on pregnenolone synthesis. Cycloheximide also blocked AII stimulation of pregnenolone synthesis in intact cells. We propose that protein synthesis is required for angiotensin to exert its stimulatory effects at one particular locus: activation of mitochondrial pregnenolone synthesis. Protein synthesis is not required for other angiotensin-stimulated processes in bovine adrenal glomerulosa cells.