Rheumatoid arthritis (RA) is a chronic disease characterized by inflammation and destruction of joints and the surrounding tissue (1;2). This destruction is mediated through autoimmune processes as evidenced by the appearance of specific serologic markers, including rheumatoid factor and anti-citrullinated protein antibodies (ACPA) (3–6). ACPA develop years before disease onset, but are remarkably disease specific, suggesting an important etiologic role for immune recognition of self-proteins modified by citrullination (7;8). RA susceptibility in general and the appearance of ACPA in particular are linked to a limited subset of HLA-DR haplotypes, including DRB1*04:01 (DR0401), implying that recognition of self-peptides by CD4+ T cells is important in driving antibody responses in RA and also suggesting that some T cell epitopes could be citrullinated (1;9). CD4+ T cells that respond to citrullinated peptides (cit-specific T cells) have been implicated in the progression of arthritis in several murine models - both indirectly through T cell help and by direct infiltration into the joint (10;11). T cells from RA patients have been shown to expand and secrete cytokines in response to stimulation in vitro with citrullinated peptides (12–14). However these studies did not characterize cit-specific T cells directly ex vivo, leaving unanswered questions about the magnitude and phenotype of T cell responses to citrullinated antigens in human subjects. Previous studies of T cells in autoimmunity have predominantly relied on assays that include in vitro manipulation, which therefore have a limited ability to examine T cell frequency and potentially skew T cell phenotype as a result of that manipulation. Direct ex vivo analysis, while preferable, has been a technical challenge due to the inherently low frequencies of epitope specific CD4+ T cells (15). In this paper we sought to visualize and characterize CD4+ T cell responses to citrullinated antigens directly ex vivo utilizing a panel of DR0401 tetramers. We developed this tetramer panel by first identifying a set of citrullinated peptides derived from synovial antigens with the capacity to bind HLA-DR0401, and then confirmed in vivo immune responsiveness using HLA-DR0401 transgenic mice. The corresponding DR0401 tetramers were able to detect cit-specific T cells in the peripheral blood of RA patients and healthy controls. However, in patients as compared to controls, the frequency of cit-specific T cells was increased, as was the percentage of these cells displaying a Th1 memory phenotype. Frequency of cit-specific T cells varied significantly with disease duration and with treatment using biologic therapies. Thus, we have directly identified cit-specific T cells from RA patients that are likely active in the pathogenesis of RA.