ABCA3 is highly expressed at the membrane of lamellar bodies in alveolar type II cells, in which pulmonary surfactant is stored. ABCA3 gene mutations cause fatal surfactant deficiency in newborns. We established HEK293 cells stably expressing human ABCA3 and analyzed the function. Exogenously expressed ABCA3 is glycosylated and localized at the intracellular vesicle membrane. ABCA3 is efficiently photoaffinity labeled by 8-azido-[α³²P]ATP, but not by 8-azido-[γ³²P]ATP, when the membrane fraction is incubated in the presence of orthovanadate. Photoaffinity labeling of ABCA3 shows unique metal ion-dependence and is largely reduced by membrane pretreatment with 5% methyl-β-cyclodextrin, which depletes cholesterol. Electron micrographs show that HEK293/hABCA3 cells contain multivesicular, lamellar body-like structures, which do not exist in HEK293 host cells. Some fuzzy components such as lipids accumulate in the vesicles. These results suggest that ABCA3 shows ATPase activity, which is induced by lipids, and may be involved in the biogenesis of lamellar body-like structures.