We combined several recent technological advances in immunology and molecular biology to identify and sequence a large number of T-cell receptor (TCR) genes specific for a particular antigen. We utilized class II MHC tetramers and interferon-gamma surface capture to isolate from samples of peripheral blood the population of CD4(+) T cells responding to a peptide derived from influenza hemagglutinin and restricted by HLA-DR1. Detailed analysis of hundreds of clones from three different patients revealed an extremely diverse repertoire, with little overlap between patients. We observed no dominant usage of particular Vbeta segments nor any clear CDR3 sequence motif in the responding T cells, but most of the clones appear to utilize acidic residues in the CDR1 and CDR3 regions, presumably to interact with the exposed basic residues in the MHC-peptide complex. This methodology could be expanded to a large scale to identify the generalized rules governing TCR-MHC engagement and factors which shape the T-cell repertoire after vaccination and in autoimmune pathologies.