Striking examples of T cell cross-reactivity have been reported for CTL responses to viruses [17, 20] . It was shown that CTLs that were elicited during a primary viral infection might also respond when the same mice are re-infected with unrelated viruses. By mapping the different viral epitopes to which a particular T cell clone can respond, it was demonstrated that these cross-reactive epitopes can share very little sequence identity [17, 20] leading to the conclusion that CTLs are extremely non-specific [8, 17, 20] . Reviewing the literature, we compiled a set of 19 cross-reactive epitopes in Table 1 . These epitopes are restricted to the K b , K d , D b , HLA-A1, HLA-A2, and HLA-B62 MHC alleles. Some of the epitopes share only a few amino acids; one is even different on all positions, while others share the majority of the amino acids. We assumed that the first epitope (x) in a cross-reactive pair (x,y) is the original epitope for which the cross-reactive CTL clone was first raised, and that it was observed to respond to y later (see Table 1 ). To test whether these cross-reactive epitopes that differ markedly in their sequence could nevertheless have structurally similar amino acids on the non-identical positions, we did the following. First we computed the similarity of the cross-reactive epitopes S O . Then we constructed an ensemble of random peptides that have the same identical positions as the cross-reactive epitope pair but otherwise consist of random amino acids (see Materials and Methods for details). We then computed the baseline (or the expected) peptide-similarity as the average random similarity denoted S E . In 16 out of 19 pairs the observed similarity S O exceeded the expected baseline similarity S E (see Table 1 and Fig. 3A, p,0 .02, Fisher's exact test). Fig. 3A shows the observed (S O ) versus the baseline expected similarity (S E ) and the solid line presents the case where S O = S E . This plot demonstrates that crossreactive epitopes are significantly more similar than unrelated peptides with the same level of sequence identity. Thus, in crossreacting T cell ligands non-identical positions are significantly more conservative than random. Fig. 3B shows this more explicitly. The 19 epitope pairs were split in two groups according to the level of sequence identity; less than 50% and larger than or equal to 50% identity. For both groups we compute the percent excess observed similarity of the cross-reactive constituents defined as 100?(S O 2S E )/S E From Fig. 3 , we clearly see that for ''seemingly'' unrelated sequences (identity,50%) the excess observed similarity (y-axis) is on average 25.8% +/2 10.8%, i.e., when sequence identity is low, the observed similarity is much higher than the expected similarity. Conversely, for epitopes sharing more than half the amino acids, excess similarity drops markedly (2.0% +/2 4.3%) probably because cross-reactivity is maintained by the more numerous identical positions. The difference in excess observed similarity between the groups is highly significant (p,0.001, rank test), which suggests that amino acid identity is a poor measure for estimating physicochemical similarity, and thus T cell crossreactivity. In summary, the above results demonstrate that biochemical similarity plays a large role in defining CTL crossreactivity when sequence identity is low. In such cases, crossreactivity is observed for non-identical, but conservative, substitutions preserving structural and/or physiochemical properties satisfying the idiosyncratic binding constrains of the responding TCR. The columns are as follows: 1) MHC restriction, 2) source pathogen and protein for initial infection, 3 