SummaryHuman leukocytes were stimulated in vitro with peptides corresponding in sequence to the highly variable helix of the a1 domain of various HLA-B and -C molecules. A CD4'CD8 -cytotoxic T cell line, CTLAV, that is specific for the HLA-B7 peptide presented by HLA-DR11 .1 was obtained. The HLA-DR11.2 molecule, which only differs at three residues from HLA-DR11 .1, did not present the HLA-B7 peptide to CTLAV Peptides from the a1 domain helix of other HLA-A and HLA-B molecules, but not HLA-C molecules, competed with the HLA-B7 peptide for binding to HLA-DR11.1. A cell line (WT50) that coexpresses HLA-B7 and HLA-DR11 .1 was killed by CTLAV in the absence ofany added HLAB7 peptide. The processing and presentation ofHLAB7 in these cells appears to be through the endogenous, and not the exogenous, pathway of antigen presentation. Thus, Brefeldin A inhibits presentation and chloroquine does not . Furthermore, introduction of purified HLA-B7 molecules into HLA-DR11 .1', HLA-B7 -cells by cytoplasmic loading via osmotic lysis of pinosomes, but not by simple incubation, rendered them susceptible to CTLAV killing. These results provide an example of class II major histocompatibility complex (MHC) presentation of a constitutively synthesized self protein that uses the endogenous pathway ofantigen presentation . They also emphasize the capacity for presentation of MHC peptides by MHC molecules.