The mode of action of methotrexate (MTX) in rheumatoid arthritis (RA) is unknown. The hypothesis that its cytostatic effect may be involved has been questioned based on the evidence of several negative results, the most intriguing being its lack of effect on the Lymphoblastic Transformation Test (LTT) and other lymphoproliferations in cultures from patients' samples. Our study demonstrates that LTT evaluation by 3H-thymidine uptake, a standard method, is misleading when applied to MTX-treated cells. At in-vitro concentrations similar to those present in the red blood cells of RA patients, MTX produced an early block in the cell cycle without reducing the cellular uptake of 3H-thymidine. While the explanation of this discrepancy is still open to discussion, it is clear that future studies on the immunological status of RA patients on MTX should not use thymidine uptake for the measurement of the lymphocyte response to mitogens and various stimuli, but must rely on other methods for evaluating DNA synthesis.