C yclosporine (CsA) is both a clinical immunosuppressive drug and a probe to dissect intracellular signaling pathways. In vitro, CsA inhibits lymphocyte gene activation by inhibiting the phosphatase activity of calcineurin (CN). In clinical use, CsA treatment inhibits 50-75% of CN activity in circulating leukocytes. We modeled this degree of CN inhibition in primary human leukocytes in vitro in order to study the effect of partial CN inhibition on the downstream signaling events that lead to gene activation. In CsA-treated leukocytes stimulated by calcium ionophore, the degree of reduction in CN activity was accompanied by a similar degree of inhibition of each event tested: dephosphorylation of nuclear factor of activated T cell proteins, nuclear DNA binding, activation of a transfected reporter gene construct, IFN-gamma and IL-2 mRNA accumulation, and IFN-gamma production. Furthermore, the degree of CN inhibition was reflected by a similar degree of reduction in lymphocyte proliferation and IFN-gamma production in the allogeneic mixed lymphocyte cultures. These data support the conclusion that CN activity is rate-limiting for the activation of primary human T lymphocytes. Thus, the reduction of CN activity observed in CsA-treated patients is accompanied by a similar degree of reduction in lymphocyte gene activation, and accounts for the immunosuppression observed.