Lipid rafts on the cell surface are believed to be very important as platforms for various cellular functions. The aim of this study was to know whether defective lipid efflux may influence lipid rafts on the cell surface and their related cellular functions. We investigated macrophages with defective lipid efflux from ATP binding cassette transporter A1-deficient (Abca1-KO) mice. Lipid rafts were evaluated by the following two novel probes: a biotinylated and protease (subtilisin Carlsberg)-nicked derivative of theta-toxin and a fluorescein ester of polyethylene glycol-derived cholesterol. Lipid rafts in Abca1-KO macrophages were increased, as demonstrated by both probes. Moreover, activities of nuclear factor kB, mRNA and intracellular distribution, and secretion of tumor necrosis factor-alpha (TNF-alpha) were examined after stimulation by lipopolysaccharides (LPSs). LPS-induced responses of the activation of nuclear factor kB and TNF-alpha were more prompt and accelerated in the Abca1-KO macrophages compared with wild-type macrophages. Modification of lipid rafts by cyclodextrin and nystatin corrected the abnormal response, suggesting an association between the increased lipid rafts and abnormal TNF-alpha secretion. We report here that Abca1-KO macrophages with defective lipid efflux exhibited increased lipid rafts on the cell surface and accelerated TNF-a secretion. - Koseki, M., K- i. Hirano, D. Masuda, C. Ikegami, M. Tanaka, A. Ota, J. C. Sandoval, Y. NakagawaToyama, S. B. Sato, T. Kobayashi, Y. Shimada, Y. OhnoIwashita, F. Matsuura, I. Shimomura, and S. Yamashita. Increased lipid rafts and accelerated lipopolysaccharide-induced tumor necrosis factor-a secretion in Abca1-deficient macrophages.