Improved understanding of the immune events discriminating between seropositive arthralgia and clinical synovitis is of key importance in rheumatology research. Ample evidence suggests a role for Th17 cells in rheumatoid arthritis. We hypothesized that CD4+CD161+ cells representing Th17 lineage cells may be modulated prior to or after development of clinical synovitis. Therefore, in a cross-sectional study, we investigated the occurrence of CD4+CD161+ T-cells in seropositive arthralgia patients who are at risk for developing rheumatoid arthritis and in newly diagnosed rheumatoid arthritis patients. In a prospective study, we evaluated the effect of methotrexate treatment on circulating CD4+CD161+ T-cells. Next, we assessed if these cells can be detected at the level of the RA joints. Precursor Th17 lineage cells bearing CD161 were found to be increased in seropositive arthralgia patients. In contrast, circulating CD4+CD161+ T-cells were decreased in newly diagnosed rheumatoid arthritis patients. The decrease in CD4+CD161+ T-cells correlated inversely with C-reactive protein and with the 66 swollen joint count. Methotrexate treatment led to normalization of CD4+CD161+ T-cells and reduced disease activity. CD4+CD161+ T cells were readily detected in synovial tissues from both early and late-stage rheumatoid arthritis. In addition, synovial fluid from late-stage disease was found to be enriched for CD4+CD161+ T-cells. Notably, synovial fluid accumulated CD4+CD161+ T-cells showed skewing towards the Th1 phenotype as evidenced by increased interferon-gamma expression. The changes in peripheral numbers of CD4+CD161+ T-cells in seropositive arthralgia and early rheumatoid arthritis and the enrichment of these cells at the level of the joint predict a role for CD4+CD161+ T-cells in the early immune events leading to clinical synovitis. Our findings may add to the development of RA prediction models and provide opportunities for early intervention.