Studies in human beings, animals, and cell systems show that the rate-limiting step in steroidogenesis is the conversion of cholesterol to pregnenolone. In the adrenals and gonads, this step is subject to both acute and chronic regulation. Chronic regulation is primarily, but not exclusively at the level of gene transcription, leading to the production of more steroidogenic machinery and thus increasing the cellular capacity for steroidogenesis. Chronic regulation can be inhibited by inhibiting protein synthesis with cycloheximide, but this response varies among various cell types and species. Although the P450scc enzyme system that converts cholesterol to pregnenolone is inherently very slow, the principal site of acute regulation is at the delivery of free cholesterol to mitochondria, rather than at the delivery of reducing equivalents to P450scc. Even when the Vmax of the P450scc system is increased 6-fold by genetic engineering, delivery of cholesterol to the enzyme remains rate-limiting. Targeting of a genetically engineered fusion of the P450scc system to either mitochondria or to the endoplasmic reticulum of non-steroidogenic cells demonstrates that the mitochondrial environment is absolutely required for the conversion of cholesterol to pregnenolone, and that this absolute requirement is not based on either the nature of the available electron donors for P450scc or the availability of substrate. Various factors have been proposed as the essential mediator for the transport of cholesterol into mitochondria to initiate steroidogenesis. A recently identified protein termed Steroidogenic Acute Regulatory protein (StAR) has the necessary properties of enhancing steroidogenesis, rapid cAMP inducibility and rapid cycloheximide sensitivity that characterize the long-sought acute regulator of steroidogenesis. StAR is expressed in steroidogenic tissues exhibiting an acute response but not in steroidogenesis. StAR is expressed in steroidogenic tissues exhibiting an acute response but not in steroidogenic tissues (placenta, brain) that do not exhibit this response. Mutations in StAR are now shown to cause Congenital Lipoid Adrenal Hyperplasia, the last unsolved form of CAH. The actions of StAR can be circumvented by the use of hydroxycholesterols that can freely diffuse into mitochondria, proving that StAR functions as an acute regulator of cholesterol access to mitochondria.