Calcineurin (CaN), a Ca2+/calmodulin-dependent serine/threonine phosphatase, has been shown to be inhibited by the complex of the immunosuppressant cyclosporin A (CsA) and its receptor, cyclophilin (CyP), but not by either alone. In the current study, the topological relationship between cyclophilin and the subunits of CaN has been explored using chemical cross-linking agents. In the presence of cyclosporin, 125I-CyP, shown to bind to CsA, is extensively cross-linked to the B subunit of CaN but not the catalytic A subunit. However, the A subunit is required for binding of the CyP.CsA complex, since cross-linking to recombinant B subunit alone does not occur. The kinetics of association indicate a saturable reaction with a Kd of less than 70 nM. Cross-linking to the B subunit occurs with cross-linkers that span from 0 to 16 A and employ different cross-linking chemistry, indicating direct contact between the B subunit and CyP. Similar cross-linking to the B subunit has been observed with the complex of 125I-labeled FK506 binding protein (FKBP) and FK506 but not with the FKBP-rapamycin complex. CyP.CsA cross-linking to CaN is Ca2+/calmodulin-dependent with intact CaN, but Ca2+/calmodulin-independent after digestion to remove the calmodulin binding and autoinhibitory domain.