—The role of apolipoprotein A-IV (apoA-IV) in lipoprotein metabolism has not been established. The aim of the present study was to investigate the role of apoA-IV in reverse cholesterol transport by comparing cellular cholesterol efflux to serum or serum fractions from control mice and from mice transgenic for human apoA-IV (HuA-IVTg mice). When Fu5AH hepatoma cells were used, the cholesterol efflux to serum from either control or transgenic mice was similar. When control J774 macrophage cells were used, a comparison of efflux to serum or lipoprotein-deficient serum (LPDS) failed to demonstrate any differences between control and transgenic mice. In contrast, when the J774 cells were pretreated with cAMP, there was a stimulation of efflux to whole serum or LPDS from HuA-IVTg mice. cAMP treatment had no effect on efflux to serum or LPDS from control mice. Pretreatment of the cells with cAMP did not enhance the efflux response to high density lipoprotein isolated from HuA-IVTg mouse serum. Our results suggest that apoA-IV, unassociated with high density lipoprotein particles, is responsible for enhanced cholesterol efflux. This study illustrates the role of lipid-free apolipoproteins in mediating cellular cholesterol efflux with use of a biological fluid and is potentially of physiological relevance, especially in apolipoprotein-rich extravascular fluids.