Methotrexate (MTX) is an effective treatment for RA and its effects may be partly due to cytokine modulation. Herein, we assessed the effects of a single MTX dose on the production and circulating concentrations of several cytokines and soluble receptors in 42 RA patients on three consecutive days. Three patient groups were studied: (a) 16 patients taking the first MTX dose, (b) 11 patients on chronic MTX treatment and (c) a control group of 15 patients not treated with MTX. Cytokine production was studied in peripheral blood mononuclear cells (PBMNC) and in a whole-blood culture system (WBCS). Group (a) had a more active disease according to laboratory parameters as well as higher circulating IL-6 levels (P = 0.002). The secretion of IL-1 beta by stimulated PBMNC (P = 0.008) was higher in this group and decreased significantly (P = 0.03) after a single MTX dose. No significant change in any parameter was observed after MTX in group (b). In the total patient group, circulating concentrations of IL-1 beta and TNF-alpha were low but blood cells showed a high capacity of production for these cytokines. In contrast for sTNFRs, high circulating levels but a limited in vitro production were observed. In conclusion, a single MTX dose may result in decreased production of IL-1 beta by PBMNC in patients with active RA. Furthermore, we observed an imbalance in the production of TNF-alpha and sTNFRs by peripheral blood cells of RA patients and propose that the WBCS is convenient for studying cytokine production in RA.