Recent evidence suggests that NPC1L1 (Niemann-Pick C1-like 1) is critical for intestinal sterol absorption in mice, yet mechanisms by which NPC1L1 regulates cellular sterol transport are lacking. In the study we used a McArdle-RH7777 rat hepatoma cell line stably expressing NPC1L1 to examine the sterol-specificity and directionality of NPC1L1-mediated sterol transport. As previously described, cholesterol-depletion-driven recycling of NPC1L1 to the cell surface facilitates cellular uptake of non-esterified (free) cholesterol. However, it has no impact on the uptake of esterified cholesterol, indicating free sterol specificity. Interestingly, the endocytic recycling of NPC1L1 was also without effect on beta-sitosterol uptake, indicating that NPC1L1 can differentiate between free sterols of animal and plant origin in hepatoma cells. Furthermore, NPC1L1-driven free cholesterol transport was unidirectional, since cellular cholesterol efflux to apolipoprotein A-I, high-density lipoprotein or serum was unaffected by NPC1L1 expression or localization. Additionally, NPC1L1 facilitates mass non-esterified-cholesterol uptake only when it is located on the cell surface and not when it resides intracellularly. Finally, NPC1L1-dependent cholesterol uptake required adequate intracellular K(+), yet did not rely on intracellular Ca(2+), the cytoskeleton or signalling downstream of protein kinase A, protein kinase C or pertussis-toxin-sensitive G-protein-coupled receptors. Collectively, these findings support the notion that NPC1L1 can selectively recognize non-esterified cholesterol and promote its unidirectional transport into hepatoma cells.