Seven A2-binding peptides were tested by the HLA class I alpha-chain refolding assay previously described for their direct binding to HLA class I alpha chains derived from a panel of 18 HLA-homozygous B-cell lines of various HLA specificities, including four A2 subtypes: A*0201, A*0204, A*0205, and A*0206. All but one test peptide possessed the major anchor residue motifs, L-V, L-L, or I-L, of A2(A*0201)/A2(A*0205)-binding peptides or the closely related motifs, I-V or V-V. This cell panel analysis confirmed the high A2 allele specificity of the test peptides, but also revealed the existence of a broad cross-binding within the A2 subgroup. Most peptides bound to the alpha chains of the A2 subtypes tested, although their binding patterns showed differences. Furthermore, the A2-binding peptides carrying the I-V or V-V motif were found to cross-react also outside of the A2 subtypes, probably with A24, A26, A28, and A29. Other A-locus allelic products, A1, A3, A11, A30, and A31, and the B-locus allelic products carried by the cells tested were essentially negative, although a few exceptions were seen.