Quantitating the frequency of T cell cross-reactivity to unrelated peptides is essential to understanding T cell responses in infectious and autoimmune diseases. Here we used 15 mouse or human CD8+ T cell clones (11 antiviral, 4 anti-self) in conjunction with a large library of defined synthetic peptides to examine nearly 30,000 TCR-peptide MHC class I interactions for cross-reactions. We identified a single cross-reaction consisting of an anti-self TCR recognizing a poxvirus peptide at relatively low sensitivity. We failed to identify any cross-reactions between the synthetic peptides in the panel and polyclonal CD8+ T cells raised to viral or alloantigens. These findings provide the best estimate to date of the frequency of T cell cross-reactivity to unrelated peptides (∼ 1/30,000), explaining why cross-reactions between unrelated pathogens are infrequently encountered and providing a critical parameter for understanding the scope of self-tolerance.