X-band electron spin resonance spectroscopy was used to investigate the binding of Mn 2 + to the apo-forms of calcineurin and its A and B subunits. The results indicated the presence of 2 Mn 2 + binding sites of different affinities (20 μmol/L and 60 μmol/L) in the calcineurin A subunit and 4 Mn 2 + binding sites in the calcineurin subunit B, 2 high affinity and 2 low affinity binding sites withK d 's of 4 μmol/L and 90 μmol/L, respectively. Interestingly and quite surprisingly, Mn 2 + binding to the holoenzyme was characterized by only 2 binding sites with K d 's of 7 μmol/L and 33 μmol/L. However, in the presence of calmodulin about 10 Mn 2 + sites were detected, and the Mn 2 + calmodulin-calcineurin complex exhibited enzymatic activity. These results, based on direct spectral measurements of the metal ligand, demonstrate that Mn 2 + binds to both free subunits of calcineurin in a manner distinct from binding to the holoenzyme. Also, the data suggest that conformational changes occur upon heterodimer formation and association of the holoenzyme with the regulatory protein calmodulin.