Rat adrenocortical cell suspensions (10(6) cells) were incubated with ACTH (40 nM) in 2 ml of Krebs-Ringer bicarbonate buffer for 90 min. About 42 nmol of corticosterone and 14 nmol of 18-hydroxydeoxycorticosterone were generated and released into the medium. Aminoglutethimide at 50 microM inhibited the steroidogenesis to 16%. Mitochondrial pellets were prepared from the cells incubated in the absence, or in the presence, of ACTH and aminoglutethimide, and cholesterol content was determined. The mitochondria of the cells incubated without the drugs contained 25.2 micrograms cholesterol/mg protein. Cholesterol content increased by 10% in the mitochondria of the ACTH-stimulated cells. The mitochondria of the cells incubated in the presence of both ACTH and aminoglutethimide contained 143% of cholesterol compared to those of the nontreated cells. When rats were subjected to ether stress after aminoglutethimide pretreatment, cholesterol content of the mitochondrial fraction increased to about 200% compared to that of the control rats. These results suggest that a cholesterol pool exists in the adrenocortical mitochondria and that the amount increases during the steroidogenic stimulation of the cells. The mitochondria were fixed with filipin-containing fixative and examined by freeze-fracture electron microscopy. Accumulations of filipin-cholesterol complexes were observed in the inner membrane of the mitochondria as protuberances or pits 25 nm in diameter.