A fluoroimmunoassay employing europium-streptavidin and time-resolved fluorimetry was used to measure binding of biotin-labeled peptides to H-2Dk molecules. A fluorescein-labeled octameric peptide from the middle T (MT) protein of mouse polyoma virus was identified that specifically binds to purified Dk using a previously-established assay based on size exclusion chromatography. A europium immunoassay was adapted to measure binding of a biotin-derivative of this peptide to purified Dk. The assay was found to be sensitive and highly specific. Binding was optimal at pH 5.0 and 24-27 degrees C, and it was not enhanced in the presence of additional beta2-microglobulin (beta2m). Excellent results were also obtained in experiments with fixed cells that express Dk. This assay is expected to be useful for high volume, routine analysis of peptide binding to MHC class I molecules.