Cyclosporine (CsA) blocks T cell responses in vitro by inhibiting the phosphatase activity of calcineurin (CN) and thus preventing the activation of cytokine transcription. In the study presented here, we measured the extent of inhibition of these functions in the tissues of CsA-fed mice. Mice fed increasing doses of CsA were assessed for CsA blood and tissue levels, spleen cell CN activity, ex vivo spleen cell cytokine induction by A23187, and in vivo interferon-gamma induction during an allogeneic response. The CN activity of spleen homogenates and cell suspensions and the ex vivo cytokine responses of spleen cells from CsA-treated mice were inhibited with a 50% inhibitory concentration (IC50) greater than 300 microg/L. The in vivo interferon-gamma response to an allogeneic ascites tumor was also inhibited by CsA treatment, with IC50s between 517 and 886 microg/L. The true IC50 for CsA in vivo may be even higher, as CsA levels in spleen and kidney were 4-fold higher than concomitant blood levels. We conclude that inhibition of CN activity by systemically administered CsA leads to a parallel reduction in cytokine gene induction in response to an allogeneic stimulus. In light of our previous clinical findings that therapeutic levels of CsA in renal transplant patients were associated with only partial inhibition of CN activity, these current results support the concept that partial CN inhibition can account for both the immunosuppression and the immunocompetence of CsA-treated patients.