Antigen-driven development of persistent self-reactive Th cells underlies the chronic, progressive nature of many autoimmune diseases. It is crucial to understand the behavior of these self-reactive Th cells; however, they have been notoriously difficult to isolate directly ex vivo. Collagen-induced arthritis (CIA) can be initiated in I-A(q)-expressing mice (DBA/1) using heterologous type II collagen (cII) immunization and is dependent on Th cells that are specific for a single immunodominant epitope. Here, we identify one compartment of cII-specific Th cell using TCR beta expression, cell surface phenotype, and direct single-cell repertoire analysis. A subpopulation of CD4(+)V beta 10(+) T cells up-regulates both CD44 and GL7 and expands significantly in response to initial priming in the majority of animals (D9: 70%). The cII-specific V beta 10(+) primary responders are further resolved through expression of a highly restricted junctional region, previously associated with autoimmune disease. This cII-specific clonotype rapidly re-expands upon antigen recall and can be isolated from the lymph nodes of arthritic animals. These single-cell analyses quantify the emergence, decline and rapid re-emergence of a self-reactive Th cell population in vivo and outline one strategy for isolating these cells directly ex vivo.