We have previously reported that activation of murine T cell hybridomas leads to expression of Fas (CD95) and its ligand (FasL) which subsequently interact, even on the same cell, leading to apoptotic cell death. Since the immunosuppressive drugs cyclosporin A (CsA) and FK506 block activation-induced apoptosis in T cell hybridomas, we examined whether such compounds affect cell death by interfering with expression of Fas, FasL or both, or whether they block Fas signal transduction. We have found that CsA- and FK506-treated cells did not exhibit transcription of FasL mRNA after activation and were lacking functional FasL protein on their surface as determined by staining and the ability to induce apoptosis in Fas+ target cells. In contrast, no inhibition of the elevated Fas mRNA expression was observed in cells activated in the presence of CsA or FK506. Surprisingly, however, cell surface Fas levels were consistently lower on cells activated in the presence of immunosuppressive drugs than on activated cells, suggesting Fas expression is regulated at several levels. Nevertheless, cells activated in the presence of CsA or FK506 underwent apoptosis upon treatment with anti-Fas antibody, while unactivated cells did not. Furthermore, CsA and FK506 do not interfere with Fas signaling since anti-Fas induced apoptosis in Fas+ target cells was unaffected by these drugs. We therefore conclude that CsA and FK506 block activation-induced apoptosis in T cell hybridomas predominantly by interfering with activation signals leading to FasL expression and, further, that the regulation of the expression of Fas and FasL on activated T cells is differentially controlled.