The ABC transporter ABCA1 has been implicated to control cholesterol efflux in a variety of cell types including macrophages, fibroblasts, and intestinal epithelial cells. In this study we have investigated whether the 6-kD protein anionic peptide fraction/calcium binding protein (APF/CBP) which has homology to apolipoprotein A1 may regulate efflux mediated by lipoproteins. APF/CBP was purified from T-tube bile by ultracentrifugation and preparative reversed phase HPLC. Cholesterol efflux to a variety of acceptors was determined using cultured fibroblasts from controls and patients with Tangiers disease. APF/CBP (0.1 to 2.4 mug/ml) inhibited ApoA-1 (2 mug/ml) mediated cholesterol efflux from normal fibroblasts in a dose dependent manner but had no effect on aspecific efflux to methyl-beta -cyclodextrin or phosphatidylcholine liposomes. In ABCA1 deficient fibroblasts no effect of APF/CBP on efflux was seen. We conclude that APF/CBP specifically interferes with ApoA-1 mediated cholesterol trafficking. We hypothesize that competitive binding to ABCA1 may explain the decreased ApoA-1 mediated efflux from fibroblasts.