Sterol carrier protein-2 (SCP-2), also known as nonspecific lipid transfer protein, plays a crucial role in intracellular lipid trafficking and metabolism. Numerous studies have shown that SCP-2 greatly enhances the translocation rate of cholesterol (Ch) and various phospholipids (PL) between membrane bilayers. Our group recently showed that human recombinant SCP-2 (13.2 kDa) can also accelerate intermembrane transfer of Ch hydroperoxide and PL hydroperoxide species. Whether SCP-2 actually binds Ch or PLs during facilitated translocation has been controversial. The interaction has been assessed by others, using fluorescence titration techniques with fluorophore-tagged Ch or PLs. However, reported binding constants vary widely for any given lipid (e.g. up to 100-fold for Ch), and could be suspect due to the relatively massive attached fluorophore group. To circumvent these problems, we have devised a novel approach for measuring binding, based on genetically appending a biotinylated peptide to the SCP-2 C-terminus for use in surface plasmon resonance (SPR) measurements. Biotinylated SCP-2 was immobilized on a streptavidincoated chip and the on-rates, off-rates and binding constants for various lipids, including oleic acid, linoleic acid, and the 5α-, 6α-, and 7α-hydroperoxides of Ch were assessed by SPR, using Biacore™ instrumentation. These studies demonstrate that the SPR approach is not only practicable, but clearly superior to previously used approaches. Assessing SCP-2 binding of lipid hydroperoxides by this technique is highly significant in the context of cell damaging oxidative stress because SCP-2 trafficking of these species under stress conditions could greatly expand their range of prooxidant and stress signaling activity. (Supported by NIH grant CA72630)