It is becoming increasingly accepted that removal of cellular cholesterol occurs by at least two pathways, one involving the well-described aqueous diffusion mechanism and another promoted by lipid-free apolipoproteins. We compared the contribution of apolipoprotein-dependent and -independent pathways, taking into consideration the influence of cellular metabolism, on cholesterol efflux promoted by different extracellular acceptor types. The acceptors used were assumed to participate in only passive efflux by lipid-dependent mechanisms (phospholipid vesicles and trypsin-modified high density lipoproteins) or to stimulate efflux by apolipoprotein-dependent pathways (purified apolipoprotein A-I and high density lipoproteins). Apolipoprotein-mediated cholesterol efflux was only apparent in growth-arrested or cholesterol-enriched cells and required metabolic energy. In contrast, cholesterol efflux by apolipoprotein-depleted acceptors did not depend on cell growth state, cholesterol enrichment, or metabolic energy. Apolipoprotein-mediated efflux was not observed at temperatures below 22 degrees C, while apolipoprotein-independent efflux was only reduced by 50% at 4 degrees C compared with incubations at 37 degrees C. Additionally, apolipoproteins promoted a more rapid and larger decrease in intracellular cholesteryl esters when measured by changes in cholesteryl ester radioactivity, mass, or the pool of cholesterol available for esterification by acyl coenzyme A:cholesterol acyltransferase. Efflux of excess cellular cholesterol by an apolipoprotein-dependent pathway appears to involve specific cellular events consistent with the properties of an active transport pathway and distinguishable from cholesterol efflux by apolipoprotein-depleted acceptors through passive mechanisms.