We have used antigen-specific T-cell clones plus antigen to examine the biochemical effects of leflunomide on T cells during an immune response. Leflunomide inhibited both antigen and IL-2-stimulated proliferation of these clones, with an ED50 of 8-10 μM for either stimuli. Conversely, antigen-stimulated cytokine production and up-regulation of the IL-2 receptor (CD25) were relatively insensitive to inhibition by leflunomide. IL-2-receptor-mediated signaling, however, was inhibited by this drug. Using32P-orthophosphate metabolic labeling and anti-phosphotyrosine immunoprecipitation, we were able to show that leflunomide inhibited IL-2-stimulated tyrosine kinase activity. Furthermore, we demonstrated that leflunomide directly inhibited the IL-2 stimulated tyrosine kinase activity of p56lck. These data suggest that inhibition of IL-2-stimulated p56lck activity could play a role in leflunomide-mediated immunosuppression.