Efflux of [14C]cholesterol from various cells was monitored in the presence of discoidal complexes of egg phosphatidylcholine and purified apolipoproteins, containing either apoAI, AII, or Cs. Particles containing apoAI were more efficient acceptors than those containing apoAII or Cs when the donor cells were J774 macrophages. No differences were observed when the same acceptor preparations were exposed to Fu5AH rat hepatoma or rabbit aortic smooth muscle cells. The differential efficiency of apolipoproteins in stimulating cholesterol removal from J774 cells was maintained in a plasma membrane-enriched fraction isolated from the same cells. Nonlinear regression analysis of kinetic data obtained from J774 cells exposed to apoAI complexes indicated that cholesterol efflux was best fitted to a curve describing the release from two kinetic compartments. Approximately 10% of cholesterol was transferred from a rapidly exchangeable pool with a t1/2 ranging between 1.5 and 3 h, and the remaining fraction was released from a slower pool with a t1/2 of about 20 h. Modulation of cholesterol efflux from J774 cells by either varying the concentration or the apolipoprotein composition of the acceptors influenced the size of the pools and the t1/2 of the slow pool. Kinetics of cholesterol efflux from membranes isolated from J774 cells also best fit a two-compartment model and modification of the apolipoprotein composition of the acceptor induced a pattern of changes in pool size and half-time similar to that described for whole cells. In the three cell lines studied, we consistently resolved a slow pool with a half-time ranging between 15 and 20 h. In smooth muscle cells only the slow pool was evident, whereas in Fu5AH a very large fast pool was also resolved. In contrast to J774 cells, apolipoprotein composition of the acceptor did not influence the pools in these two cell lines. These results led us to propose a new model regarding the influence of multiple kinetic pools of cholesterol on the regulation of cholesterol desorption from the cell membrane.