The immunosuppressive drugs cyclosporin A (CsA) and FK506 both interfere with a Ca(2+)-sensitive T-cell signal transduction pathway, thereby preventing the activation of specific transcription factors (such as NF-AT and NF-IL2A) involved in lymphokine gene expression. CsA and FK506 seem to act by interaction with their cognate intracellular receptors, cyclophilin and FKBP, respectively (see ref. 11 for review). The Ca2+/calmodulin-regulated phosphatase calcineurin is a major target of drug-isomerase complexes in vitro. We have therefore tested the hypothesis that this interaction is responsible for the in vivo effects of CsA/FK506. We report here that overexpression of calcineurin in Jurkat cells renders them more resistant to the effects of CsA and FK506 and augments both NFAT- and NFIL2A-dependent transcription. These results identify calcineurin as a key enzyme in the T-cell signal transduction cascade and provide biological evidence to support the notion that the interaction of drug-isomerase complexes with calcineurin underlies the molecular basis of CsA/FK506-mediated immunosuppression.