The calcium/calmodulin-regulated phosphatase calcineurin (CN) is the site of action of the immunosuppressive drugs cyclosporin A (CsA) and FK506. CN has recently been established as a key signaling enzyme in the T cell signal transduction cascade and an important regulator of transcription factors such as NF-AT and OAP/Oct-1, which are involved in the expression of a number of important T cell early genes. CsA and FK506 act by forming complexes with their respective intracellular receptors cyclophilin and FKBP (immunophilins), which can then bind to CN, inhibiting its enzymatic activity and thereby preventing early gene expression. CN is comprised of two subunits: a 59-kDa catalytic subunit (CNA), which contains a calmodulin binding domain and autoinhibitory region, and a 19-kDa intrinsic calcium binding regulatory subunit (CNB). In this study, we have utilized a series of deletion mutants of the CNA subunit to investigate the subunit and molecular requirements that govern the interaction of CN with drug-immunophilin complexes. The calmodulin binding and autoinhibitory domains of the CNA subunit were found to be dispensable for the binding of CN to drug-immunophilin complexes. In contrast, we found that the regulatory CNB subunit appears to play an obligatory role in this interaction and have defined an amino acid sequence of the CNA subunit which forms the binding site for CNB. Although necessary, the CNB subunit per se is not sufficient to mediate an interaction with drug-immunophilin complexes; amino acid residues of the CNA subunit, specifically a region located within the putative catalytic domain, are also required for the interaction of CN with both FKBP-FK506 and cyclophilin A-CsA.