Functioning bovine adrenocortical cells in monolayer culture were shown to obtain cholesterol for steroid synthesis from plasma low density lipoprotein (LDL). When grown in medium devoid of lipoproteins, the cells developed a minimal enhancement in steroid secretion in response to ACTH or cholera toxin. However, when LDL was available, steroid secretion was stimulated 4- to 9-fold. To determine the mechanism for this effect, we used LDL in which the protein component was labeled with 125I and the cholesteryl ester component was labeled with [3H]cholesteryl linoleate. These studies demonstrated that the cells derived cholesterol from LDL by binding the lipoprotein at a high affinity receptor site, internalizing it, and hydrolyzing its cholesteryl esters within lysosomes. The resultant free cholesterol was used for steroid synthesis and also acted to suppress the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase and cholesterol synthesis within the cell. LDL receptor activity was enhanced several-fold by treatment of the cells with ACTH or cholera toxin. High density lipoprotein, which did not bind to the LDL receptor, was not degraded with high affinity by the cells and did not support steroid synthesis. The current data suggest that the bovine adrenal cortex can obtain cholesterol for steroid hormone secretion from circulating LDL by means of a high affinity LDL receptor pathway. In a subsequent paper in this series, a similar high affinity LDL-binding site is demonstrated in membranes prepared from fresh bovine adrenocortical tissue.