According to recent crystallographic studies, the TCR-αβ contacts MHC class I-bound antigenic peptides via the polymorphic V gene-encoded complementarity-determining region 1β (CDR1β) and the hypervariable (D) J-encoded CDR3β and CDR3α domains. To evaluate directly the relative importance of CDR1β polymorphism on the fine specificity of T cell responses in vivo, we have taken advantage of congenic Vβ a and Vβ b mouse strains that differ by a CDR1 polymorphism in the Vβ10 gene segment. The Vβ10-restricted CD8+ T cell response to a defined immunodominant epitope was dramatically reduced in Vβ a compared with Vβ b mice, as measured either by the expansion of Vβ10+ cells or by the binding of MHC-peptide tetramers. These data indicate that Vβ polymorphism has an important impact on TCR-ligand binding in vivo, presumably by modifying the affinity of CDR1β-peptide interactions.