Calcineurin (CN), a heterodimer composed of a catalytic subunit, calcineurin A (CNA) and regulatory subunit, calcineurin B (CNB), is involved in many cellular processes. We investigated the denaturation of CNA by urea in the presence or absence of CNB and found that CNB protected CNA against urea. The phosphatase activity of CNA that had been exposed to low urea concentrations (below 4 M), in the presence CNB, was higher than that of the separately urea-treated subunits mixed just prior to assay. In order to analyze the protection of CNA by CNB, we investigated the K(m) and V(max), and intrinsic fluorescence, of CNA that had been exposed to various concentrations of urea in the presence or absence of CNB. CN had an increased V(max) and decreased K(m) when exposed to 1 to 2 M urea. In addition, the kinetic parameters and intensity of intrinsic fluorescence of the AB complex and isolated subunits were quite different in 3 M urea. These results indicate that CNB not only plays an important role in regulating CNA, but also protects it against denaturation by urea.