Although it was recently recognized that sterol carrier protein-2 (SCP-2) interacts with fatty acids, little is known regarding the specificity of SCP-2 for long-chain fatty acids or branched-chain fatty-acid-like molecules. Likewise the location of the fatty-acid binding site within SCP-2 is unresolved. A fluorescent cis-parinaric acid displacement assay was used to show that SCP-2 optimally interacted with 14-22 carbon chain lipidic molecules: polyunsaturated fatty acids > monounsaturated, saturated > branched-chain isoprenoids > branched-chain phytol-derived fatty acids. In contrast, the other major fatty-acid binding protein in liver, fatty-acid binding protein (L-FABP), displayed a much narrower carbon chain preference in general: polyunsaturated fatty acids > branched-chain phytol-derived fatty acids > 14- and 16-carbon saturated > branched-chain isoprenoids. However, both SCP-2 and L-FABP displayed a very similar unsaturated fatty-acid specificity profile. The presence and location of the SCP-2 lipid binding site were investigated by fluorescence energy transfer. The distance between the SCP-2 Trp50 and bound cis-parinaric acid was determined to be 40 A. Thus, the SCP-2 fatty-acid binding site appeared to be located on the opposite side of the SCP-2 Trp50. These findings not only contribute to our understanding of the SCP-2 ligand binding site but also provide evidence suggesting a potential role for SCP-2 and/or L-FABP in metabolism of branched-chain fatty acids and isoprenoids.