Alternatively, ABCG4 may have differential mechanisms of substrate recognition and transport compared with ABCA1 and ABCG1. PrebHDL formed by ABCA1 contains relatively more PC and less SM compared with HDL formed by ABCG1. The SM-poor preb-HDL could be a good acceptor of cholesterol and SM transported by ABCG1. ABCG4, expressed in the central nervous system, would mediate the efflux of cholesterol to the PC-rich SP600125 apoE-HDL formed by ABCA1. The different substrates of ABCA1, ABCG1, and ABCG4 may be important for the production of HDL composed of various lipids. Furthermore, the contents of cholesterol, PC, and SM in HDL may be regulated by the balanced efflux mediated by ABCA1, ABCG1, and ABCG4. We have shown that lipid effluxes mediated by ABCA1 and ABCG1 are increased and impaired, respectively, by a decrease of cellular SM levels. In this study, we showed that the efflux of cholesterol mediated by ABCG4 is not affected by decreased SM levels. The distinct localizations of ABCA1, ABCG1, and ABCG4 on the plasma membrane may account for the different effects of cellular SM levels on their functions. A membrane environment of lipid rafts that are thick and liquid-ordered might be required for the function of ABCG1. Decreased SM levels disrupt raft domains, where ABCG1 resides, leading to the decreased activity of ABCG1. On the other hand, a decrease of SM level would be favorable for ABCA1 because it enlarges the area where ABCA1 functions. ABCG4 is localized to Brij 96 raft domains and partly to non-raft domains as shown in Fig. 2. This unsettled localization of ABCG4 may explain the observation that decreased SM levels had no effect on its activity. It has been reported that cellular SM content is increased in macrophages treated with acetyl-low density lipoprotein, in macrophages during differentiation from monocyte, in peritoneal macrophages during aging of rats, and in alveolar macrophages during postnatal development. Changes in cellular SM levels may be involved in regulating the function of ABCA1 and ABCG1, and in the development of atherosclerosis. Mendez et al. have reported that apoA-I removes cellular cholesterol from Triton X-100-soluble membranes, and that HDL removes cholesterol from Triton X-100-soluble and -insoluble membranes. These findings are in accord with our result that ABCA1 and ABCG1 were localized to Triton X-100-soluble and insoluble domains, respectively. We suggest that apoA-I removes cholesterol from non-raft domains, where ABCA1 resides, and that HDL removes cholesterol both from raft domains, where ABCG1 resides, and from non-raft domains, possibly by simple diffusion. Because cholesterol, newly synthesized or derived from lipoproteins like LDL, is trafficked to raft domains.