Dilauryl fluorescein (DLF) is a lipid soluble molecule that becomes fluorescent when lauric acid is removed by hydrolysis The purpose of these studies was to evaluate DLF as a potential probe for the function of reconstituted high-density lipoproteins (rHDL) as hydrophobic drug transport vehicles. The DLF containing rHDL nanoparticles were characterized regarding their physical/chemical properties, including molecular diameter, molecular weight, chemical composition, and buoyant density. We investigated the uptake of DLF from rHDL in cells that overexpress the scavenger receptor (SR-B1), known to facilitate the selective cellular uptake of cholesteryl esters from HDL. These studies show that DLF can be incorporated into rHDL and redistributed in the plasma compartment. In addition, these studies demonstrated an enhanced uptake and hydrolysis of DLF from rHDL by cells that overexpress the SR-B1 receptor, suggesting the involvement of a receptor mediated mechanism. The incorporation of DLF into the rHDL nanoparticles appear to protect against hydrolysis in the systemic circulation based on the lower rate of rHDL/DLF hydrolysis compared with the free DLF during incubation with human plasma. DLF may thus be used as a probe to track the movement and metabolism of HDL core constituents, including cancer chemotherapeutic agents.