Molecules encoded by a single major histocompatibility complex class I gene can associate with any one of a large number of peptide ligands. T-cell receptors have the capacity to discriminate among these peptide-class I complexes and in many cases bind only a single peptide-class I complex with sufficient affinity to trigger effector function. In contrast, it is generally assumed that class I-specific alloantibodies are indifferent to peptide heterogeneity, being directed toward allele-specific determinants on the molecule. In this report, three monoclonal antibodies were used to precipitate Kb molecules from cell lysates. Surprisingly, in each case a different set of peptides was found to be associated with Kb as detected by peptide-dependent Kb-specific alloreactive cytolytic T lymphocytes or by biochemical resolution. These results demonstrate that the affinity of binding by alloantibodies can be affected by the endogenous peptide ligand.