Research on CD8 T cell-mediated inflammatory diseases requires a better understanding of target epitopes and the constraints placed upon these by major histocompatibility complex (MHC) class I binding restrictions, especially those that relate to predisposing alleles. We used linear trap quadrupole fourier transform (LTQ-FT) tandem mass spectrometry to identify naturally processed and presented peptides eluted from the MHC-negative myeloid leukaemia cell line K562 transfected with specific MHC class I genes. We provide information on the peptidome of HLA-B*39:06, which is associated with the autoimmune disease type 1 diabetes, and extend the analysis to include a further five human leukocyte antigen (HLA) alleles (HLA-A*02:01/-A*11:01/-A*24:02/-B*18:01/-B*38:01) studied under identical experimental conditions. We identified a total of 3095 individual peptides with a mascot score ≥40 (HLA-A*02:01 = 569 peptides, -A*11:01 = 904, A*24:02 = 257, -B*18:01 = 615, -B*38:01 = 453, -B*39:06 = 297). Peptides had a preferential length of nine amino acids and originated mainly from cytoplasmic or nuclear proteins. Eluted peptides revealed a strong binding motif with binding anchor positions at position 2 (P2) and the C-terminus (PΩ). Peptides eluted from HLA-A*02:01 showed a P2 preference for leucine (62% of total peptides have Leu at P2) and PΩ preference for valine (49%). Similar data are provided for HLA-A*11:01 (P2:Thr, 29%; PΩ:Lys, 49%), -A*24:02 (P2:Tyr, 78%; PΩ:Phe, 41%), -B*18:01 (P2:Glu, 77%; PΩ:Tyr, 32%), -B*38:01 (P2:His, 51%; PΩ:Leu, 45%) and -B*39:06 (P2:Arg/His, 24%; PΩ:Ala, 64%). This work thus gives an overview of the naturally processed and presented repertoire of several common and autoimmune disease-related HLA alleles, which may be useful in studying autoreactive CD8 T cell responses and the role of HLA in disease susceptibility.