Although sterol carrier protein-2 (SCP-2) stimulates sterol transfer in vitro, almost nothing is known regarding the identity of the putative cholesterol binding site. Furthermore, the interrelationship(s) between this SCP-2 ligand binding site and the recently reported SCP-2 long chain fatty acid (LCFA) and long chain fatty acyl-CoA (LCFA-CoA) binding site(s) remains to be established. In the present work, two SCP-2 ligand binding sites were identified. First, both [4-(13)C]cholesterol and 22-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3beta-ol (NBD-cholesterol) binding assays were consistent with a single cholesterol binding site in SCP-2. This ligand binding site had high affinity for NBD-cholesterol, K(d) = 4.15 +/- 0.71 nM. (13)C NMR-labeled ligand competition studies demonstrated that the SCP-2 high affinity cholesterol binding site also bound LCFA or LCFA-CoA. However, only the LCFA-CoA was able to effectively displace the SCP-2-bound [4-(13)C]cholesterol. Thus, the ligand affinities at this SCP-2 binding site were in the relative order cholesterol = LCFA-CoA > LCFA. Second, (13)C NMR studies demonstrated the presence of another ligand binding site on SCP-2 that bound either LCFA or LCFA-CoA but not cholesterol. Photon correlation spectroscopy was consistent with SCP-2 being monomeric in both liganded and unliganded states. In summary, both (13)C NMR and fluorescence techniques demonstrated for the first time that SCP-2 had a single high affinity binding site that bound cholesterol, LCFA, or LCFA-CoA. Furthermore, results with (13)C NMR supported the presence of a second SCP-2 ligand binding site that bound either LCFA or LCFA-CoA but not cholesterol. These data contribute to our understanding of a role for SCP-2 in both cellular cholesterol and LCFA metabolism.