Cellular cholesterol homeostasis is regulated by oxygenated cholesterol metabolites called oxysterols. While the importance of oxysterols in the acute regulation of cholesterol homeostasis is known, the precise molecular mechanisms through which oxysterols exert their effects remain to be elucidated. LY295427 (1) is a known antagonist of the cholesterol-homeostatic effects of 25-hydroxycholesterol (25-HC), a biologically active oxysterol. In order to examine the mechanism of action of this antagonism, and to further explore recent evidence suggesting that the membrane effects of 25-HC contribute to acute cholesterol regulation, we synthesized the enantiomer of LY295427 (ent-LY295427). ent-LY295427 (2) will serve as a unique probe to provide insight into the role of transcription-independent mechanisms in regulation of cholesterol homeostasis.