Previously, using an oxysterol to induce cholesterol trafficking to the Endoplasmic Reticulum (ER), we reported a dissociation between cholesterol transport to two important cholesterol regulatory components in the ER: the cholesterol esterifying enzyme ACAT (Acyl CoA:Cholesterol Acyltransferase) and the membrane-bound transcription factor SREBP (Sterol Regulatory Element Binding Protein) (X. Du, Y.H. Pham and A.J. Brown, Effects of 25-hydroxycholesterol on cholesterol esterification and SREBP processing are dissociable: implications for cholesterol movement to the regulatory pool in the endoplasmic reticulum, J. Biol Chem. 279 (2004) 47010-47016). Here, we employed low-density lipoprotein (LDL) as a more physiologically-relevant mode of cholesterol delivery, and compared cholesterol transport to ACAT (determined by esterification) and SREBP (assessed by processing) in mutant Chinese Hamster Ovary cells that have cholesterol-trafficking defects (including Niemann-Pick type C). We showed clear differences in kinetics between the two, with impaired cholesterol trafficking to SREBP being resolved more rapidly than to ACAT. This is unlikely to be due to a reduced threshold of cholesterol sensed by the SREBP system relative to ACAT, since both responded to LDL-derived cholesterol within 2 h whereas the divergence observed between the two was prolonged (>20 h). Furthermore, ACAT inhibition did not expand the ER regulatory pool of cholesterol as judged by unaltered sensitivity of SREBP processing to LDL. Collectively, our data favor the contention that there are different cholesterol pools and/or transport pathways which feed ACAT and SREBP within the ER.