Calcineurin (CaN) is a Ca2+/calmodulin-dependent protein phosphatase found in brain and other tissues. It is a heterodimer consisting of a catalytic subunit (CaN-A) and a Ca(2+)-binding regulatory subunit (CaN-B). The primary structure of CaN-B indicates that it, like calmodulin, is an EF-hand protein and binds four Ca2+ ions. Eu3+, due to its favorable spectroscopic and chemical properties, has been substituted for Ca2+ in CaN-B to determine the metal ion-binding properties of this "calmodulin-like" protein. Excitation of the 7F0-->5D0 transition of Eu3+ results in a spectrum similar to that of calmodulin, consisting of three peaks. Analysis of the spectral titration curves reveals four Eu(3+)-binding sites in CaN-B. The affinities vary: sites I and II have dissociation constants of 1.0 +/- 0.2 and 1.6 +/- 0.4 microM, respectively; the values for sites III and IV are Kd = 140 +/- 20 and Kd = 20 +/- 10 nM, respectively. Binding of Tb3+ is slightly weaker. Tb3+ luminescence, sensitized by tyrosine, reveals that for lanthanides the highest affinity sites lie in the C-terminal domain. Energy transfer distance measurements between Eu3+ and Nd3+ in sites III and IV reveal a separation of 10.5 +/- 0.5 A, which suggests that these sites are arranged in a typical EF-hand pair. This information indicates that the overall structure of CaN-B is similar to the dumbbell-shaped proteins troponin-C and calmodulin, but is more like TnC in its metal-binding properties.