alpha-Tocopherol (alphaTocH) is transported in association with lipoproteins in the aqueous milieu of the plasma. Although up to 50% of circulating alphaTocH is transported by high-density lipoproteins (HDLs), little is known about the mechanisms of uptake of HDL-associated alphaTocH. During the current study, human apolipoprotein (apo)E-free HDL subclass 3 (HDL3) labelled with [14C]alphaTocH was used to investigate uptake mechanisms of HDL3-associated alphaTocH by a permanent hepatoblastoma cell line (HepG2). HDL3-associated alphaTocH was taken up independently of HDL3 holoparticles in excess of apoA-I comparable with the non-endocytotic delivery of cholesteryl esters to cells termed the 'selective' cholesteryl ester uptake pathway. Experiments with unlabelled HDL3 demonstrated net mass transfer of alphaTocH to HepG2 cells. Time-dependent studies with [14C]alphaTocH-labelled HDL3 revealed tracer uptake in 80-fold excess of apoA-I and in 4-fold excess of cholesteryl linoleate. In addition to HLDs, low-density lipoprotein (LDL)-associated alphaTocH was also taken up in excess of holoparticles, although to a lesser extent. These findings were confirmed with unlabelled lipoprotein preparations, in which HDL3 displayed a 2- to 3-fold higher alphaTocH donor efficiency than LDLs (lipoproteins adjusted for equal amounts of alphaTocH). An important factor affecting particle-independent uptake of alphaTocH was the cellular cholesterol content (a 2-fold increase in cellular cholesterol levels resulted in a 2.3-fold decrease in uptake). Pulse-chase studies demonstrated that some of the HDL3-associated alphaTocH taken up independently of holoparticle uptake was resecreted along with a newly synthesized apoB-containing lipoprotein fraction.