OBJECTIVE: Rheumatoid arthritis (RA)-associated autoantibodies include those directed at the kinase site of BRAF (v-Raf murine sarcoma viral oncogene homolog B1), a serine-threonine kinase involved in the MAPK signaling pathway. To understand anti-BRAF immunization, we sought to identify BRAF mutations in the peripheral blood lymphocytes (PBLs) of patients with RA. METHODS: We first cloned the major BRAF region known for mutations in the pCR2.1 vector, using genomic DNA from the PBLs of 8 RA patients. For each patient, 100 clones were sequenced. In 5 of 8 patients, we detected a new BRAF mutation in 1 clone. The frequency of this new mutation was evaluated by droplet digital polymerase chain reaction in PBLs from RA patients and controls. To test whether p.Val600Ala influences the kinase activity of BRAF, we developed an in vitro assay based on phosphorylation of MEK-1, a major BRAF substrate. RESULTS: A BRAF mutation, p.Val600Ala, was identified in 1 of 8,000 PBLs and 1 of 6,000 T lymphocytes from RA patients and in 1 of 12,500 PBLs and 1 of 12,500 T lymphocytes from controls. The BRAF p.Val600Ala mutation was not correlated with the presence of anti-BRAF autoantibodies. The p.Val600Ala mutation activated phosphorylation of MEK-1 in vitro. CONCLUSION: Most RA patients have a p.Val600Ala mutation in the BRAF gene. This mutation activates the kinase activity of BRAF. The p.Val600Ala mutation could activate the MAPK pathway, leading to the activation of T lymphocytes.