Methotrexate (MTX) is an anti-folate drug used in cancer chemotherapy because of its anti-proliferative effects. However, it is unclear whether the anti-proliferative effects of MTX contribute to the efficacy of low-dose MTX in the treatment of rheumatoid arthritis (RA). To date, either no change or a paradoxical increase in lectin-induced proliferation has been observed in cultures of peripheral blood mononuclear cells (PBMC) from MTX-treated RA patients (RA+MTX). In these earlier studies, high folate-containing media and tritiated thymidine (3H-TdR) were used. Our studies were designed to test the hypothesis that the use of a culture medium with a low folate content along with tritiated deoxyuridine (3H-UdR) permits detection of diminished phytohemagglutinin (PHA)-induced proliferative responses of PBMC from RA+MTX. The data demonstrate decreased PHA-induced cellular proliferation of cultured PBMC from RA+MTX compared with controls. When comparing the PBMC proliferative responses in high vs low folate medium, a significantly greater increase (P<0.05) in proliferation occurs in the cells from RA+MTX cultured in the high folate medium. This suggests that an in vivo folate-deficient state of the cells from RA+MTX may be corrected in vitro when a high folate medium is used in culture. We conclude that the use of3H-UdR and a medium containing folate within the normal range of plasma folate levels eliminates artifacts associated with the use of high folate medium and3H-TdR, which obscures the anti-proliferative effect of MTX in RA patients. © 1990 Springer-Verlag.