Two activities have been identified for the immunosuppressive metabolite of leflunomide, A77 1726: inhibition of dihydroorotate dehydrogenase (DHO-DHase), an enzyme involved in the biosynthesis of pyrimidine nucleotides (PyN); and inhibition of protein tyrosine kinases. The in vitro potency of A77 1726 as a DHO-DHase inhibitor is reported to be 10- to 500-fold greater than as a tyrosine kinase inhibitor. These observations suggested that the immunosuppressive efficacy of leflunomide in vivo is related to inhibition of DHO-DHase. However, observations that patients with disorders in the PyN synthetic pathway are not overtly immunodeficient militate against this hypothesis. We investigated the effects of leflunomide in vivo and report that amelioration of lymphoproliferative and autoimmune diseases in MRL/MpJ-lpr/lpr (lpr/lpr) mice by leflunomide is not accompanied by reduced PyN concentrations in lymph node cells. Our hypothesis that lymphocytes could salvage serum uridine to counter the effects of reduced PyN synthesis in vivo was supported by in vitro studies. Finally, we observed that amelioration of disease correlated with a reduction of tyrosine phosphorylated proteins in lymph node cells of lpr/lpr mice. These observations suggest that the primary mechanism by which leflunomide prevents autoimmune and lymphoproliferative diseases in lpr/lpr mice is not depletion of PyN, but correlates with reduced tyrosine phosphorylation concentrations in lymph node cells.