The ability of sterol carrier protein-2 (SCP-2) to interact with long chain fatty acyl-CoAs was examined. SCP-2 bound fluorescent fatty acyl-CoAs at a single site with high affinity. Kd values for cis- and trans-parinaroyl-CoA were 4.5 and 2.8 nM, respectively. Saturated 10-18-carbon and unsaturated 14-20-carbon fatty acyl-CoAs displaced SCP-2-bound fluorescent ligand. Oleoyl-CoA and oleic acid (but not coenzyme A) significantly altered SCP-2 Trp50 emission and anisotropy decay, thereby increasing SCP-2 rotational correlation time, SCP-2 hydrodynamic radius, and SCP-2 Trp50 remaining anisotropy up to 1.7-, 1.2-, and 1.3-fold, respectively. These changes were not accompanied by significant alterations in protein secondary structure as determined by circular dichroism. Finally, SCP-2 differentially altered the fluorescence emission and anisotropy decays of bound cis- and trans-parinaroyl-CoA. Both fluorescent fatty acyl-CoAs were located within a very ordered (limited cone angle of rotation) environment within SCP-2, as shown by a remaining anisotropy of 0.365 and 0.361 and a wobbling cone angle of 12 and 13 degrees , respectively. These anisotropy values were very close to those of such ligands in a propylene glass. However, the rotational relaxation times exhibited by SCP-2-bound cis- and trans-parinaroyl-CoA, 8.4-8.8 ns, were longer than those for the corresponding free fatty acid, 7.5-6.6 ns. These data show for the first time that SCP-2 is a fatty acyl-CoA-binding protein.