ACTH produced a 75% increase in pregnenolone biosynthesis from endogenous precursors in isolated cells prepared from the rat Snell adrenocortical carcinoma 494. On the addition of 24- and 25-hydroxycholesterol to the tumor cells, the rate of pregnenolone synthesis increased 10-fold but was insensitive to the presence of ACTH. Addition of lipoprotein cholesterol resulted in increased pregnenolone biosynthesis when ACTH was present. High density lipoprotein cholesterol appeared to be internalized and used for steroidogenesis preferentially to low density lipoprotein cholesterol. The cholesterol ester hydrolase activity of the cytosolic fraction of the tumor was found to be extremely low compared to that of the normal adrenal cell. These results, noting also the low cholesterol content of the tumor cells, suggested that the lack of availability of cholesterol was the factor responsible for the poor steroidogenic response of the cells to ACTH. The major steroid product of the tumor cells was determined to be deoxycorticosterone. This correlated with the low levels of steroid 11-beta-hydroxylase activity detected in the adrenal tumor mitochondria compared to the mitochondrial cholesterol desmolase activity. Little of the mitochondrial cytochrome P-450 appeared to function in a steroid 11-beta-hydroxylase complex.