Cholesterol is among one of the most decorated molecules in biology today, the study of which produced not only a number of Nobel Prizes but also key findings that influence our study of physiology and medicine today. It is the single starting substrate of the steroidogenic pathway, the synthesis of steroid hormones such as progesterone, testosterone, and corticosterone, which control a variety of essential physiological and metabolic functions including salt and water balance, spermatogenesis, follicular development and maintenance of pregnancy. Steroidogenesis is a unique process in which all of the steroid hormones are derived from a single substrate, cholesterol, by the CYP11A1 enzyme located on the matrix side of the inner mitochondrial membrane. Steroidogenesis is rapidly triggered in response to induction by tropic hormones such as ACTH or LH binding to their respective G-protein coupled receptors on the plasma membrane, triggering a rise in intracellular cAMP. This process, however, requires that cholesterol be trafficked across the aqueous intermembrane space, a substantial obstacle for the steroidogenic cell due to cholesterol’s extreme hydrophobicity. My studies provide evidence that this intramitochondrial trafficking is facilitated by the steroidogenic acute regulatory protein (STAR) and not the translocator protein (TSPO). My studies conclusively eliminate TSPO from our current models of the steroidogenic process and demonstrate that a TSPO ligand that has been shown to reduce progesterone synthesis is, in fact, an inhibitor of 3b-HSD, a downstream enzyme of progesterone synthesis. During my studies, I also generated STAR knockout MA-10 Leydig cell lines, the first in vitro model of STAR deletion in a steroidogenic cell line, providing evidence that induction of STAR expression is required for the steroidogenic process and that this role cannot be compensated for by TSPO. My studies also demonstrate that extracellular cholesterol sources, such as serum lipoproteins, can contribute to the total pool of cholesterol substrate utilized by the cell to synthesize steroid hormones. I also characterize a group of hCG-responsive MA-10 cells, an improved model system for the ongoing study of steroidogenic pathways beyond the mitochondria. I also provide evidence that changes in ER cholesterol synthesis in MA-10 cells can influence expression of the STAR protein, pointing toward the possible involvement of the sterol regulatory element binding protein (SREBP) family of transcription factors. These studies set the stage for the study of cholesterol trafficking pathways beyond the mitochondria and characterize models that can be used as a tool to study cholesterol sourcing and trafficking proteins that support steroidogenesis.