The interaction of cyclosporin (A) and cyclosporin derivatives with cyclophilins (A, B), and (C) has been investigated by means of fluorescence measurement techniques. Since Trp-121 of cyclophilin (A) is in close contact with bound cyclosporins and changes its fluorescence emission upon binding, direct estimation of (K_{d}) values for cyclosporins is straightforward in this case. Cyclophilins (B) and (C), however, display no evident binding-dependent fluorescence changes suitable for the estimation of their binding affinities. This problem can be circumvented by measuring the variations of fluorescence emission intensities of a mixture of cyclophilin (A) and the fluorescence measurements unsuitable for cyclosporin binder as a function of ligand concentration. Application of a mixed-mode kinetic analysis then allows the calculation of the cyclosporin binding affinity of the second binder in the system. The dissociation constant for cyclosporin (A) /cyclophilin (A) was found to be (36.8 \mathrm{~nm}). Mixed-mode kinetic calculations yielded (K_{d}) values of 9.8 and (90.8 \mathrm{~nm}) for cyclophilins (B) and (C), respectively. The analysis was extended to noncyclophilin (weak) cyclosporin binders such as calmodulin and actin, resulting in approximate (\boldsymbol{K}{d}) values of 1.2 and (5.7 \mu \mathrm{M}), respectively. Using the same approach, the (K{d}) values of a series of different cyclosporin derivatives were determined. 1994 Academic Press, Inc.