Various cytokines and reactive oxygen species (ROS) play a fundamental role in the inflammatory and immunologic processes of rheumatoid arthritis (RA). Methotrexate (MTX) is one of the disease-modifying anti-rheumatic drugs and its effect may be partly due to the modulation of immunologic or inflammatory reactions by some cytokines. In the present study, we investigated the effects of MTX on the gene expression and synthesis of interleukin-6 (IL-6), and the proliferative activity and the production of ROS in the fibroblast-like synoviocytes (FLSs) obtained from the patient of RA. The expression or production of IL-6 was induced spontaneously, and augmented by the addition of recombinant human IL-6 or recombinant human IL-1 beta and TNF-alpha in FLSs. These spontaneous and augmented IL-6 expressions or productions were suppressed by treatment with low-concentration of MTX (1 microg/ml). Also, IL-6 stimulated the proliferation of FLSs, and this IL-6 driven proliferation was inhibited with the treatment of MTX or N-acetylcysteine (NAC, 1 mM). Furthermore, ROS production in FLSs was increased significantly by IL-6, and its effect was also abrogated in the presence of MTX or NAC. These results suggest that inflammatory reaction in the synovium of RA patients could be augmented by the autocrine or other cytokine-induced production of IL-6 with subsequent generation of ROS in the synoviocytes, and the modulations of IL-6 synthesis and ROS production may contribute to the therapeutic effects of MTX for RA.