The mode of action of low-dose methotrexate (MTX) in rheumatoid arthritis (RA) is unclear. The effects of MTX are mediated primarily through inhibition of dihydrofolate reductase, resulting in a dose-dependent inhibition of purine and pyrimidine synthesis. Other folate-dependent metabolic pathways might be secondarily affected. One such pathway is the regeneration of methionine from homocysteine, with subsequent formation of the methyl donor S-adenosylmethionine (SAM) and polyamines, which are important in cell-mediated immune reactions. To assess whether MTX inhibits SAM and polyamine synthesis in lymphocytes, pokeweed mitogen-stimulated mononuclear cells from healthy donors were incubated with MTX. This resulted in decreased proliferation and IgG, IgM, and IgM rheumatoid factor synthesis. However, addition of folinic acid, methionine, SAM, or spermidine resulted in reversal of the MTX-mediated inhibition. These data suggest that MTX inhibits the folate-dependent pathway of methionine regeneration, thereby inhibiting SAM and polyamine synthesis. Since RA lymphocytes have increased concentrations of polyamines, the beneficial effects of MTX in RA may be related to its potential ability to reduce polyamine synthesis.