The regulatory pool of cholesterol is located in the endoplasmic reticulum (ER) and is key to how mammalian cells sense and respond to changes in cellular cholesterol levels. The extent of cholesterol esterification by the ER-resident protein, acyl-coenzyme A:cholesterol acyl-transferase (ACAT), has become the standard method for monitoring cholesterol transport to the ER and is assumed to reflect the regulatory pool of ER cholesterol. The oxysterol, 25-hydroxycholesterol (25HC), is thought to trigger intracellular cholesterol transport to the ER. In support of this contention, we confirmed previous reports that 25HC activates cholesterol esterification and is a potent suppressor of the sterol regulatory element-binding protein (SREBP) pathway. Processing of the ER membrane-bound SREBP into a soluble transcription factor is controlled by cholesterol levels in the ER. In this study, we addressed whether or not cholesterol esterification necessarily reflects cholesterol movement to the cholesterol homeostatic machinery in the ER as determined by SREBP processing. We found that three agents that inhibited the ability of 25HC to induce cholesterol esterification (progesterone, nigericin, and monensin) did not have a corresponding effect on 25HC suppression of SREBP processing. Moreover, ACAT inhibition did not alter the sensitivity of SREBP processing to 25HC. Therefore, cholesterol esterification by the ER-resident protein ACAT is dissociable from cholesterol transport to the cholesterol homeostatic machinery in the ER. In light of our results, we question the security of previous work that has inferred cholesterol transport to the ER regulatory pool based solely on cholesterol esterification.