To investigate type II collagen (CII)-specific CD4+ T cell receptors involving in Collagen-induced arthritis (CIA) in DBA/1J mice as a model of rheumatoid arthritis in humans, TCRVβ usage in draining lymph nodes (dLNs) was assessed by flow cytometric analysis at 3, 5, and 8 weeks after bovine CII immunizations. In the early stage of CIA, the draining lymph node CD4+ T cells from CIA mice showed a higher proportion of CD4+ Vβ3+subsets compared with those from control mice. The CD4+ Vβ3+ T cells were specifically and primarily expanded by antigen-specific stimulation in in vitro culture of dLNs lymphocytes and splenocytes from CIA mice. In addition, CII-reactive response was observed when CD4+ Vβ3+ T cells were added to a non-responding T cell population. The adoptive transfer of CD4+ Vβ3+ T cells produced exaggerated arthritis compared with that in the control group. Our results indicate that CD4+Vβ3+ T cells, which were selectively expanded in dLN of CIA mice, play a pivotal role in CIA pathogenesis.