Calcineurin (CaN) binds Ca2+-saturated calmodulin (CaM) with relatively high affinity; however, an accurate steady-state K-d value has not been determined. In this report, we describe, using steady-state and stopped-flow fluorescence techniques, the rates of association and dissociation of Ca2+-saturated CaM from CaN heterodimer (CaNA/CaNB) and CaNA only. The rate of Ca2+/CaM association was determined to be 4.6 x 10(7) M-1 s(-1). The rate of Ca2+/CaM dissociation from CaN was slower than previously reported and was approximately 0.0012 s(-1). In preparations of CaNA alone (no regulatory CaNB subunit), the dissociation rate was slowed further to 0.00026 s(-1). From these data we calculate a Kd for binding of Ca2+-saturated CaM to CaN of 28 pM. This Kd is significantly lower than previously reported estimates of similar to 1 nM and indicates that CaN is one of the highest affinity CaM-binding proteins identified to date. (c) 2005 Elsevier Inc. All rights reserved.