The colorimetric assay for 5-aminoimidazole-4-carboxamide ribotide (AICAR) transformylase (phosphoribosylaminoimidazolecarboxamide formyltransferase; EC 18.104.22.168) has been extensively modified. The modified assay is based upon the short-term permanganate oxidation of the folate product, tetrahydrofolate (H4folate) to p-aminobenzoyl glutamate (pABG). The modified assay was used to detect the transformylase activity in crude extracts of peripheral-blood mononuclear cells (PBMCs). Azathioprine and its metabolite, thioinosinic acid (tIMP), are competitive inhibitors (with respect to AICAR) of the chicken liver transformylase and the transformylase from PBMCs of the MRL/lpr mouse, an animal model of systemic autoimmune disease. The K(i) values of tIMP and azathioprine for the chicken liver enzyme are 39±4 μM and 120±10 μM, whereas the K(i) values for the enzyme from PBMCs of the MRL/lpr mouse are 110±20 μM and 90±14 μM respectively. The anti-inflammatory drugs ibuprofen and naproxen are also inhibitors of the transformylase.