Among the known mechanisms of reverse cholesterol transport (RCT), ATP binding cassette transporter G1 (ABCG1)-mediated free cholesterol (FC) transport is the most recent and least studied. Here, we have characterized the efficiencies of different acceptors using baby hamster kidney (BHK) cells transfected with human ABCG1 cDNA, which is inducible upon treatment with mifepristone. When normalized on particle number and particle surface area, the acceptor efficiency for FC efflux was as follows: small unilamellar vesicles (SUV)>LDL>reconstituted HDL>HDL2 = HDL3. Based on phospholipid content, the order was reversed. ABCG1 also mediated phospholipid efflux to human serum and HDL3. ABCG1-mediated FC efflux correlated significantly with a number of HDL subfractions and components in serum collected from 25 normolipidemic individuals: apolipoprotein A-II (apoA-II) (r2 = 0.7), apolipoprotein A-I (apoA-I) (r2 = 0.5), HDL-C (r2 = 0.4), HDL-PL (r2 = 0.4), α-2 HDL (r2 = 0.4), and preβ HDL (r2 = 0.2). ABCG1 did not enhance influx of FC or cholesteryl oleyl ether (COE) when cells were incubated with radiolabeled HDL3. ABCG1 expression did not increase the association of HDL3 with cells. Compared with control cells, ABCG1 expression significantly increased the FC pool available for efflux and the rate constant for efflux. In conclusion, composition and particle size determine the acceptor efficiency for ABCG1-mediated efflux. ABCG1 increases cell membrane FC pools and changes its rate of desorption into the aqueous phase without enhancing the association with the acceptor.