The folate status of 29 healthy control subjects, 16 rheumatoid arthritis (RA) patients taking methotrexate (MTX), and 20 RA patients who were not being treated with MTX was estimated by an assay of the folate-dependent enzymatic synthesis of serine from formate and glycine, which is termed the C1 index. Analysis of variance demonstrated that the specific activity of the enzyme system in lymphocytes was significantly lower in the MTX-treated group, with an activity approximately one-half that of the control and the non-MTX-treated groups. Since the C1 index is one of the first biochemical parameters found to be different between MTX-treated and non-MTX-treated groups, alterations in folate-mediated amino acid metabolism may be involved in the mechanism of response to MTX therapy. Use of the C1 index may assist in the development of protocols which preserve the efficacy of MTX therapy while minimizing toxicity.