LKM38 emerged as a promising cholesterol mimic because it both sustained the growth of cholesterol-auxotrophic cells and appropriately regulated key cholesterol homeostatic pathways. When presented as an ester in lipoprotein particles, LKM38 initially localized to the lysosome and subsequently trafficked to the plasma membrane and endoplasmic reticulum. LKM38 bound to diverse, established cholesterol binding proteins. Through a detailed characterization of the cellular behavior of a panel of diazirine alkyne probes using cell biological, biochemical trafficking assays and immunofluorescence approaches, we conclude that LKM38 can serve as a powerful tool for the study of cholesterol protein interactions and trafficking.[1]