ABSTRACT: All eukaryotic cells require cholesterol (Chol) for survival and regulate their steady-state levels by balancing de novo synthesis, uptake of Chol-containing lipoproteins, and Chol efflux (i.e., “Chol homeostasis”). Hereditary defects in processes that affect normal Chol homeostasis comprise a family of diseases, some of which have pathological effects on the retina. Our current understanding of Chol homeostasis in the retina remains comparatively rudimentary. Other than Chol per se, how other circulating lipids and lipid-soluble molecules impact retinal Chol homeostasis remains largely unknown and unexplored. This Supplement project piggy- backs onto an already approved parent project funded under the NIH Bench-to-Bedside initiative (NOT-OD-24- 027; Award #1167401). The overall objective of that project is to assess the effects of a novel fish oil supplement enriched in very-long-chain (C≥24) polyunsaturated fatty acids (VLCPUFAs) on cardiometabolic risk factors and visual function. The present Supplement project seeks to test the hypothesis that VLCPUFAs can alter Chol homeostasis in the retinal pigment epithelium (RPE). Relevance: the RPE, as the cellular interface between the choroidal blood supply and the neural retina, is a central player in regulating retinal Chol homeostasis. We will use cultured iPSC-derived RPE cells as a model system to test our hypothesis. We predict that exogenous VLCPUFAs will have beneficial effects on retinal Chol homeostasis in a manner that is protective against retinal degeneration. The study outcomes likely will advance our fundamental understanding of retinal Chol homeostasis and provide useful insights regarding improved therapeutic intervention against retinal degenerations.