The mechanism of action for the anti-arthritic effect of methotrexate (MTX) was investigated in rats with antigen-induced arthritis (AIA). Arthritis intensity was quantified as area under the curve (AUC) for the joint swelling. The response to MTX was in several respects similar to what is seen in the clinic. The drug reduced the AUC in a dose-dependent manner after oral weekly (2-4 mg/kg/week) or daily (0.3 mg/kg/day) dosing. This effect was not affected by supplementation with an equal dose of folate. The model thus seemed suitable for this type of study. Supplementation with folate in excess abolished the effect of MTX. A structurally similar antifolate, aminopterin, also reduced the arthritis. The effect thus seemed to be due to folate antagonism although a complete inhibition of dihydrofolate reductase (DHFR) might not be essential. Hence, it could be that the main target is a process downstream of DHFR. It has been proposed that inhibition of AICAR-transformylase induce the release of adenosine with anti-inflammatory properties. Here the adenosine antagonist R-PIA reduced the arthritis but when MTX was combined with adenosine antagonists no attenuation of the anti-arthritic effect was seen. On the contrary, three adenosine agonists (8-p-sulphophenyltheophyllamine 30 mg/kg i.p. twice daily; 3,7-dimethyl-1-propargylxanthine, p.o. 3 mg/kg/day and 8-cyclopentyl-1,3-dipropylxanthine, 1.5 mg/kg/day p.o.) potentiated MTX. The specific thymidylate synthase inhibitor 5-fluourouracil (0. 3-3.0 mg/kg/day) had no anti-arthritic effect. Neither did our data support the hypotheses that syntheses of polyamines or cytokines were primary targets. It is thus possible that the mechanism of action is inhibition of a process downstream of DHFR but the release of adenosine seems not to be important.