Although cyclosporin A (Cy A) has been widely used clinically as a potent suppressor of organ allograft rejection and has been shown to block T lymphocyte activation in vitro by inhibiting the generation of interleukin 2 (IL-2) and other lymphokines, little direct evidence is available to support the view that the immunosuppressive effects of Cy A in vivo are mediated by a similar inhibition of the autocrine lymphokine cascade. We have used a quantitative assay for the assessment of the role of the IL-2/IL-2 receptor system in the activation of the draining popliteal lymph node population after the injection of allogeneic cells in the footpad to define the effects of Cy A on the early events of lymphocyte activation in vivo and to compare them with the effects of Cy A on lymphocyte activation in vitro. The administration of Cy A in vivo had no effect on alloantigen-induced increases in cell size, percentage of cells expressing the IL-2 receptor, the spontaneous or IL-2-driven proliferation of freshly explanted cells, or the induction of cytotoxic T lymphocyte activity. These findings raise major questions about the mechanism of action of Cy A in vivo and suggest that more experimentation is required to probe the mechanisms of Cy A-induced suppression of the response to allografts.