The pool of cholesterol at the active site of the cholesterol side-chain cleavage enzyme was increased by preincubation with substrate. This process occurred at a linear time-dependent rate over 20 min, whereas uptake of cholesterol into mitochondria was complete by 1 min. Both endogenous mitochondrial cholesterol and added extramitochondrial cholesterol showed similar time-dependent substrate preincubation kinetics. The rate of metabolism of exogenous cholesterol was increased by injection of LH and decreased by treatment with cycloheximide. The rate of entry of cholesterol into the active site in both preparations, however, retained linear time-dependent kinetics. Calcium ions (2 mmol/L) increased the rate of steroidogenesis most in mitochondria preincubated for short periods and least in mitochondria preincubated for longer periods. These studies indicated that the pool of cholesterol at the active site of the enzyme can determine the rate of pregnenolone biosynthesis in the mitochondria of rat corpus luteum. Calcium ions, but not trophic hormones or cycloheximide, appeared to alter the access of cholesterol to the active site of the cholesterol side-chain cleavage enzyme during substrate preincubation.