Cyclosporin A (Sandimmun) achieves immunosuppressive activity by complex formation with cyclophilin and subsequent binding of the binary complex to and inhibiting protein phosphatase 2B (calcineurin). Complexes of nonimmunosuppressive cyclophilin binding cyclosporin analogues do not inhibit protein phosphatase 2B, suggesting a crucial role for this enzyme in T cell activation. Binding of cyclosporin A to cyclophilins A, B, and C, respectively, results in complexes of significantly different inhibitory potency. The cyclosporin molecule thus has two functional domains, one mediating cyclophilin binding and a second one endowing affinity of the complex to calcineurin, thereby inhibiting its enzyme activity. Structure-activity studies and x-ray crystallography of cyclosporin-cyclophilin complexes indicate a crucial role of leucine side chains in positions 4 and 6 of the cyclosporin macrocycle for the calcineurin interaction.