Recent crystallographic studies suggest that TCR interact with peptide/class I MHC complexes in a single preferred orientation. Although similar studies have not been performed for class II-restricted TCR, it has been proposed that T cell recognition of peptide/class II complexes has similar orientational restrictions. This study represents a functional approach to systematic analysis of this question. Twenty-one mutant A beta(d) molecules were produced by alanine scanning mutagenesis and assessed for their ability to present species variants of insulin to a panel of beef insulin-specific T cell hybridomas with limited TCR alpha- and/or beta-chain sequence differences. We demonstrate that all beef insulin-specific TCR have the same orientation on the insulin/Ad complex, such that the alpha-chain interacts with the carboxyl-terminal region of the A beta(d) alpha-helix, and the beta-chain complementarity-determining region 3 interacts with the carboxyl-terminal portion of the peptide, consistent with that observed for crystallized TCR-peptide/class I complexes. Despite this structural constraint, even TCR that share structural similarity show remarkable heterogeneity in their responses to the panel of MHC mutants. This variability appears to result from conformational changes induced by binding of the TCR to the complex and the exquisite sensitivity of the threshold for T cell activation.