OBJECTIVE To evaluate the safety and efficacy of L-asparaginase as an immunosuppressive agent in a mouse model of rheumatoid arthritis. METHODS Male DBA/1 mice with collagen-induced arthritis (CIA) were treated at different intervals with various doses of native and pegylated L-asparaginase from E. coli. The mice were observed for 4 weeks during which time arthritis was scored. Outcome parameters included effect on severity and progression of established arthritis as well as prevention of disease. In addition, X-rays from the affected joints were obtained for comparison. RESULTS Both native L-asparaginase at a dose of 50 IU/injection intraperitoneally three days a week and pegylated asparaginase (PEG-L-asparaginase) at a dose of 25 IU/injection twice a week, significantly reduced the mean arthritic score (MAS) in mice with established arthritis (p < 0.001 for PEG-L-asparaginase). When native L-asparaginase was administered before the onset of arthritis (days 14-post immunization) the number of mice developing arthritis as well as the number of arthritic paws and the severity of arthritis in the treatment group were significantly decreased (p < 0.0001). Significant differences were found in the X-ray evaluation between treated and control mice. None of the animals died due to drug related events or showed signs of asparaginase induced toxicity. CONCLUSIONS Our data provide the first direct evidence that L-asparaginase is a potent antiarthritic agent and may represent an effective second line agent for future treatment studies in juvenile and adult rheumatoid arthritis.