SummaryWe compared the conformation of empty and peptide-loaded class I major histocompatihility complex (MHC) molecules at the cell surface. Molecular conformations were analyzed by fluorescence resonance energy transfer (FRET) between fluorescent-labeled Fab fragments bound to the or2 domain of the MHC heavy chain and fluorescent-labeled Fab fragments bound to ~/2-microglobulin. No FRET was found between Fab fragments bound to empty H-2K b, but FRET was detected when empty H-2K b molecules were loaded with peptide. The magnitude of FRET depended on the sequence of the peptide used. The results imply that empty H-2K b molecules are in a relatively extended conformation, and that this conformation becomes more compact when peptide is bound. These changes, which are reflected in peptide-dependent binding of monoclonal antibodies, affect the surfaces of MHC molecules available for contact with T cell receptors and hence may influence T cell-receptor recognition of MHC molecules.