Establishment and maintenance of pregnancy depends on progesterone synthesized by luteal tissue in the ovary. Our objective was to identify the characteristics of lipid droplets (LDs) in ovarian steroidogenic cells. We hypothesized that LDs are a major feature of steroidogenic luteal cells and store cholesteryl esters. Whole bovine tissues, isolated ovarian steroidogenic cells (granulosa, theca, small luteal, and large luteal), and isolated luteal LDs were assessed for LD content, LD-associated proteins and lipid analyses. Bovine luteal tissue contained abundant lipid droplets, LD-associated perilipins 2/3/5, hormone-sensitive lipase, and 1-acylglycerol-3-phosphate O-acyltransferase ABHD5. Luteal tissue was enriched in triglycerides (TGs) compared to other tissues, except for adipose tissue. Luteal cells were distinguishable from follicular cells by the presence of LDs, LD-associated proteins, and increased TGs. Furthermore, LDs from large luteal cells were numerous and small; whereas, LDs from small luteal cells were large and less numerous. Isolated LDs contained nearly all of the TGs and cholesteryl esters present in luteal tissue. Isolated luteal LDs were composed primarily of TG, with lesser amounts of cholesteryl esters, diglyceride and other phospholipids. Bovine luteal LDs are distinct from LDs in other bovine tissues, including follicular steroidogenic cells. Luteal tissue forms in the ovary during each estrus or menstrual cycle and synthesizes progesterone, a steroid critical for early embryonic development and survival during pregnancy 1,2. Luteal tissue has a tremendous ability to synthesize progesterone, secreting up to 40 mg/day in humans 3 , and even greater quantities in cattle 4. The majority of the cholesterol utilized for progesterone biosynthesis in cattle comes from the blood in the form of high-density lipoprotein-derived cholesteryl esters with smaller amounts from low-density lipoprotein 5. Lipoproteins are internalized either through receptor-mediated endocytosis or selective cellular uptake, where cholesterol is sorted from lipoproteins within endosomes 5. Endosomal cholesterol is then believed to be trafficked to mitochondria for immediate progesterone biosynthesis or stored as cholesteryl esters in lipid reservoirs, also known as lipid droplets (LDs) for future steroid biosynthesis 5,6. In addition to its vital role in mammalian fertility, progesterone is an essential precursor of androgens, estrogens, glucocorticoids and mineralocorticoids. Therefore, the high steroidogenic output of luteal tissue allows for detailed studies of steroidogenic mechanisms, which are likely conserved among steroidogenic cell types. LDs store neutral lipids and are coated with LD-associated proteins that embed within the surrounding phospholipid monolayer. These LD-associated proteins stabilize the LD, interact with additional proteins that incorporate or remove lipids from the LD core, enable LD trafficking, and mediate association of LDs with other organelles 7. The perilipin (PLIN) proteins, designated ...