Measuring the interaction of class I human leukocyte antigens (HLA) and their peptide epitopes acts as a guide for the development of vaccines, diagnostics, and immune-based therapies. Here, we report the development of a sensitive biochemical assay that relies upon fluorescence polarization to indicate peptide interactions with recombinant soluble HLA proteins. It is a cell- and radioisotope-free assay that has the advantage of allowing the direct, real-time measurement of the ratio between free and bound peptide ligand in solution without separation steps. Peptide/HLA assay parameters were established using several HLA A*0201-specific fluorescein isothiocyanate-labeled peptides. Optimal loading of synthetic peptides into fully assembled soluble HLA-A*0201 complexes was enabled by thermal destabilization at 53 degrees C for 15 min, demonstrating that efficient peptide exchange does not require the removal of endogenous peptides from the reaction environment. An optimal ratio of three beta-2 microglobulin molecules per single HLA heavy chain was determined to maximize peptide binding. Kinetic binding studies indicate that soluble HLA-A*0201/peptide interactions are characterized by a range of moderate k(on) values (1 x 10(4) to 8.7 x 10(4) M(-1) s(-1)) and slow k(off) values (1.9 x 10(-4) to 4.3 x 10(-4) s(-1)), consistent with parameters for native HLA molecules. Testing of the A*0201-specific peptides with 48 additional class I molecules demonstrates that the unique peptide binding behavior of individual HLA molecules is maintained in the assay. This assay therefore represents a versatile tool for characterizing the binding of peptide epitopes during the development of class I HLA-based vaccines and immune therapies.