Steroid hormones play important roles in virtually every aspect of cellular metabolism, including the regulation of carbohydrate, lipid and protein metabolism and immune function (glucocorticoids), as well as salt and water balance and blood pressure regulation (mineralocorticoids). They are also critically involved in the maintenance of secondary sex characteristics, reproductive functions and muscle and bone growth (testosterone, progestins and estrogens). Thus, understanding steroid hormone production has important implications for almost every feature related to normal human health, but also to most diseases, making it quite relevant for the VA. The common precursor of steroid hormone biosynthesis is cholesterol, with the rate-limiting step being the transfer of cholesterol from the outer to the inner mitochondrial membrane. Though cholesterol trafficking for steroid hormone production has been the subject of intense investigation, the mechanisms how cholesterol traffics to the outer mitochondrial membrane remain incompletely understood. The overall goal of this proposal is to elucidate the mechanisms underlying the trafficking of cholesterol for steroidogenesis. The overall goal will be achieved by testing 2 major hypotheses. First, we propose the novel hypothesis that cholesteryl esters selectively transferred from HDL traverse the plasma membrane via SR-B1 and at the cytoplasmic side behave as micro-lipid droplets, acquiring lipid droplet- associated proteins that then direct the cholesteryl esters (micro-lipid droplets) to coalesce into mature lipid droplets or to fuse with existing cytoplasmic lipid droplets. This will be accomplished by tracing the movement of cholesteryl esters contained within HDL into lipid droplets in adrenal and gonadal cells in which specific cytosolic proteins have been knocked down or inhibited. Second, we hypothesize that the pathways through which cholesterol is transported from the plasma membrane, the endoplasmic reticulum, and lipid droplets to mitochondria each involves different mechanisms that comprise both vesicular and non- vesicular means. This aim will be accomplished using several different approaches. First, a novel cell-free in vitro reconstitution assay we have developed for cholesterol transport to mitochondria containing isolated mitochondria and recombinant StAR will be used to assess the efficiency of cholesterol delivery from the plasma membrane, the endoplasmic reticulum and lipid droplets in the presence of specific recombinant proteins, including SNAREs. Second, we will knockdown specific plasma membrane and endoplasmic reticulum proteins in granulosa and adrenal cells to assess their impact on cholesterol transport to mitochondria. Third, we will use SNARE mutant mice to compare cholesterol homeostasis and movement to mitochondria and steroidogenesis in vivo. The results from these studies should provide insights into the critical biological process of steroid hormone production.