The specificity of peptide recognition by a number of Kd-restricted CTL clones specific for HLA-CW3 or HLA-A24 was investigated. The CTL clones were derived from DBA/2 (H-2d) mice immunized with syngeneic P815 mouse cells transfected with genes encoding HLA-CW3 or HLA-A24 class I molecules. We had previously shown that CTL clones that lysed P815-CW3 transfectant target cells could lyse P815 (HLA-) target cells incubated with synthetic CW3 peptides corresponding to the COOH-terminal end of the alpha 2 domain. In the present study, we found that Kd-restricted CTL clones that lysed P815-A24 transfectant target cells recognized a synthetic peptide from the same region (residues 170-182) of the A24 molecule. CW3 and A24 differ by only one amino acid within this region. Recognition of CW3 or A24 peptides corresponded exactly with lysis of P815-HLA transfectants both for clones that mutually exclusively lysed CW3 or A24 transfectant target cells and for CW3/A24 crossreactive CTL clones. The latter CTL clones that lysed both CW3 and A24 transfectant target cells showed a clear preference for the peptide corresponding to the immunizing HLA allele. The homologous CW3 and A24 peptides could compete with each other for recognition, in contrast to a peptide from the same region of HLA-B7. Peptides from the corresponding region of the endogenous Kd and Dd/Ld molecules could also inhibit recognition of CW3 and A24 peptides. Competition with peptides apparently occurred at the level of the target cell. These results are consistent with a model whereby MHC class I molecules position protein fragments or peptides for specific recognition by T cells.