Currently, protein phosphatase 2B (calcineurin) activity is assayed based on release of [32P]phosphate from a 19-amino acid peptide (partial sequence of the regulatory subunit of cAMP-dependent protein kinase) following its [32P]ATP phosphorylation using the catalytic subunit of cAMP-dependent protein kinase. This sensitive method consumes a large amount of radioactivity and is therefore problematic as a screening method for calcineurin inhibitors. We have developed an alternative nonradioactive enzyme assay in which both phosphorylation by protein kinase and dephosphorylation by calcineurin are monitored by the simultaneous quantitative determination of phosphorylated and non-phosphorylated peptide using HPLC on an RP18 column with uv detection. The method allows the measurement of enzyme kinetics as well as the characterization of potential inhibitors. The method is comparable in sensitivity to the radioactive assay. Since calcineurin is commercially available and the substrate can be prepared in a sufficient amount, this method can be used for screening purposes. An important advantage of this new method, due to the obviation of radioactivity, is the facilitation of kinetic determinations at high substrate concentrations and the increased specificity (better identification of substrate and product). The nonradioactive substrate is very stable and can be stored for months in comparison with the 32P-peptide, which has to be freshly prepared every few weeks due to the decay of the nuclid.