We have determined a suite of crystal structures of MHC-I molecules that provide insights in the molecular basis underlying peptide binding and selection by tapasin. We demonstrate that the scoop loop of tapasin binds in the F pocket of MHC-I molecules without affecting the conformation of the rest of the peptide binding cleft, which remains empty and peptide-receptive. Furthermore, the results of our study suggest that peptides may bind first with their N termini to the peptide binding cleft and are tested thereafter for their capacities to dislodge and replace the tapasin scoop loop from the F pocket. Mutation studies revealed the importance of specific residues within the scoop loop of tapasin, with significant differences for selection between different MHC-I alleles.<\/p>\n\n\n\n