Our previous studies clearly showed that HLA-B35 and HLA-Bw53 differed only by the amino acids associated with HLA-Bw4/Bw6 epitopes, in that the former possessed Bw6 and the latter Bw4 epitope. It remains to be known whether T cell can discriminate HLA-B35 from HLA-Bw53, although the difference between these HLA antigens is discriminated by monospecific human alloantisera. To investigate allorecognition of these HLA antigens by T cells, anti-HLA-Bw53 cytotoxic T lymphocytes (CTLs) were generated. Anti-HLA-Bw53 cytotoxic T lymphocytes (CTLs) were generated. Anti-HLA-Bw53 bulk CTLs from an individual with HLA-B35 clearly discriminated HLA-Bw53 from HLA-B35. On the other hand, anti-HLA-Bw53 bulk CTLs from an individual without HLA-B35 revealed weak cross-reactivity with HLA-B35 and HLA-B51. The additional studies using HLA-Bw53 or HLA-B35-specific CTL clones showed that some but not all of the CTL clones definitively distinguish the difference between HLA-Bw53 and HLA-B35. Thus, the allospecificities formed by HLA-Bw4/Bw6 epitopes were discriminated by allogeneic T cells. The present study demonstrated that HLA-Bw4/Bw6 public epitopes play an important role in allorecognition of T cells.