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The Ca(2+)/calmodulin activated phosphatase, calcineurin, is inactivated by H2O2 or superoxide-induced oxidation, both in vivo and in vitro. However, the potential for global and/or local conformation changes occurring within calcineurin as a function of oxidative modification, that may play a role in the inactivation process, has not been examined. Here, the susceptibility of calcineurin methionine residues toward H2O2-induced oxidation were determined using a multienzyme digestion strategy coupled with capillary HPLC-electrospray ionization mass spectrometry and tandem mass spectrometry analysis. Then, regions within the protein complex that underwent significant conformational perturbation upon oxidative modification were identified by monitoring changes in the modification rates of accessible lysine residues between native and oxidized forms of calcineurin, using an amine-specific covalent labeling reagent, S,S'-dimethylthiobutanoylhydroxysuccinimide ester (DMBNHS), and tandem mass spectrometry. Importantly, methionine residues found to be highly susceptible toward oxidation, and the lysine residues exhibiting large increases in accessibility upon oxidation, were all located in calcineurin functional domains involved in Ca(2+)/CaM binding regulated calcineurin stimulation. These findings therefore provide initial support for the novel mechanistic hypothesis that oxidation-induced global and/or local conformational changes within calcineurin contribute to inactivation via (i) impairing the interaction between calcineurin A and calcineurin B, (ii) altering the low-affinity Ca(2+) binding site in calcineurin B, (iii) inhibiting calmodulin binding to calcineurin A, and/or (iv) by altering the affinity between the calcineurin A autoinhibitory domain and the catalytic center.