Although HDL-mediated cholesterol transport to the liver is well studied, cholesterol efflux from hepatocytes back to HDL is less well understood. Real-time imaging of efflux of 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino)-23,24-bisnor-5-cholen-3beta-ol (NBD-cholesterol), which is poorly esterified, and [(3)H]cholesterol, which is extensively esterified, from cultured primary hepatocytes of wild-type and sterol carrier protein-2 (SCP-2) gene-ablated mice showed that 1) NBD-cholesterol efflux was affected by the type of lipoprotein acceptor, i.e., HDL3 over HDL2; 2) NBD-cholesterol efflux was rapid (detected in 1-2 min) and resolved into fast [half time (t((1/2))) = 2.4 min, 6% of total] and slow (t((1/2)) = 26.5 min, 94% of total) pools, consistent with protein- and vesicle-mediated cholesterol transfer, respectively; 3) SCP-2 gene ablation increased efflux of NBD-cholesterol, as well as [(3)H]cholesterol, albeit less so due to competition by esterification of [(3)H]cholesterol, but not NBD-cholesterol; and 4) SCP-2 gene ablation increased initial rate (2.3-fold) and size (9.7-fold) of rapid effluxing sterol, suggesting an increased contribution of molecular cholesterol transfer. In addition, colocalization, double-immunolabeling fluorescence resonance energy transfer, and electron microscopy, as well as cross-linking coimmunoprecipitation, indicated that SCP-2 directly interacted with the HDL receptor, scavenger receptor class B type 1 (SRB1), in hepatocytes. Other membrane proteins in cholesterol efflux [SRB1 and ATP-binding cassettes (ABC) A-1, ABCG-1, ABCG-5, and ABCG-8] and several soluble/vesicle-associated proteins facilitating intracellular cholesterol trafficking (StARDs, NPCs, ORPs) were not upregulated. However, loss of SCP-2 elicited twofold upregulation of liver fatty acid-binding protein (L-FABP), a protein with lower affinity for cholesterol but higher cytosolic concentration than SCP-2. Ablation of SCP-2 and L-FABP decreased HDL-mediated NBD-cholesterol efflux. These results indicate that SCP-2 expression plays a significant role in HDL-mediated cholesterol efflux by regulating the size of rapid vs. slow cholesterol efflux pools and/or eliciting concomitant upregulation of L-FABP in cultured primary hepatocytes.