ABCA1 on the cell surface and in endosomes plays an essential role in the cell-mediated lipidation of apoA-I to form nascent HDL. Our previous studies of transgenic mice overexpressing ABCA1 suggested that ABCA1 in the liver plays a major role in regulating plasma HDL levels. The site of function of ABCA1 in the polarized hepatocyte was currently assessed by expression of an adenoviral construct encoding a human ABCA1-GFP fusion protein in the polarized hepatocyte-like WIF-B cell line. Consistent with localization of ABCA1 at the basolateral (vascular) cell surface, expression of ABCA1-GFP stimulated apoA-I mediated efflux of WIF-B cell cholesterol into the culture medium. Confocal fluorescence microscopy revealed that ABCA1-GFP was expressed solely on the basolateral surface and associated endocytic vesicles. These findings suggest an important role for hepatocyte basolateral membrane ABCA1 in the regulation of the levels of intracellular hepatic cholesterol, as well as plasma HDL.