T cell expansion typically is due to cognate interactions with specific Ag, although T cells can be experimentally activated through bystander mechanisms not involving specific Ag. TGF-β1 knockout mice exhibit a striking expansion of CD4+ T cells in the liver by 11 days of age, accompanied by CD4+ T cell-dependent necroinflammatory liver disease. To examine whether hepatic CD4+ T cell expansion in TGF-β1−/− mice is due to cognate TCR-peptide interactions, we used spectratype analysis to examine the diversity in TCR Vβ repertoires in peripheral CD4+ T cells. We reasoned that Ag-nonspecific T cell responses would yield spectratype profiles similar to those derived from control polyclonal T cell populations, whereas Ag-specific T cell responses would yield perturbed spectratype profiles. Spleen and liver CD4+ T cells from 11-day-old TGF-β1−/− mice characteristically exhibited highly perturbed nonpolyclonal distributions of TCR Vβ CDR3 lengths, indicative of Ag-driven T cell responses. We quantitatively assessed spectratype perturbation to derive a spectratype complexity score. Spectratype complexity scores were considerably higher for TGF-β1−/− CD4+ T cells than for TGF-β1+/− CD4+ T cells. TCR repertoire perturbations were apparent as early as postnatal day 3 and preceded both hepatic T cell expansion and liver damage. By contrast, TGF-β1−/− CD4+ single-positive thymocytes from 11-day-old mice exhibited normal unbiased spectratype profiles. These results indicate that CD4+ T cells in TGF-β1−/− mice are activated by and respond to self-Ags present in the periphery, and define a key role for TGF-β1 in the peripheral regulation of Ag-specific CD4+ T cell responses.