A population of CD4+ α− β+ T cells increases in the mucosal and peripheral lymphoid tissues of TCRα-chain-deficient mice with inflammatory bowel disease. The α− β+ T cells, which produce predominantly IL-4, mediate the proliferation of colonic epithelial crypts and the infiltration of large numbers of IgA-producing plasma cells into the lamina propria of the colon. To examine whether enteric Ags were recognized by a population of monoclonal α− β+ T cells leading to the intestinal inflammation, we examined the usage and clonotypes of TCR expressed by the α− β+ T cells in TCRα-chain-deficient mice with inflammatory bowel disease. Analyses of immunoprecipitates by two dimensional electrophoresis and single-cell RT-PCR revealed that TCR of the α− β+ T cells was a homodimer of β-chains that was capable of recognizing luminal bacterial Ags. PCR single-strand conformation polymorphism analysis of TCR Vβ transcripts revealed monoclonal accumulation of the α− β+ T cells in the colonic lamina propria of the diseased mice. DNA sequencing revealed the accumulation of the α− β+ T cells with the same CDR3 sequences in the colon. These findings suggest that the pathogenic CD4+ α− β+ T cells expressing a homodimeric form of the TCRβ-chains can be clonally expanded upon the stimulation with gut-derived Ags.