AbstractUsing mouse hearts from Swiss Webster mice, calcineurin was immunoprecipitated using commercially available anti‐calcineurin antibody and the resulting complex analyzed by using sodium dodecyl sulfate‐gel electrophoresis with silver staining. Distinct proteins were observed and subjected to in situ trypsin digestion followed by extraction of the resulting peptides. Peptides from each protein band were loaded onto a target for matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry and analyzed. The resulting peptide mass spectrum was compared with the Mascot and Protein Prospector databases and resulted in the specific identification of heart mitochondrial proteins, specifically Mn‐superoxide dismutase (SOD), aconitase (ACN), and malate dehydrogenase (MDH). Each of the three mitochondrial enzymes was identified with approximately 15–25% sequence coverage and all with statistical significance (P < 0.05) according to the Mascot database search engine. Tandem mass spectrometry analysis of the peptide fragmentation spectra confirmed the identification of these protein partners and also yielded the identification of mitochondrial isocitrate dehydrogenase (ICDH) as another protein in the immunoprecipitated complex. Using antibody preparations against Mn‐SOD, ACN, and ICDH showed the presence of calcineurin and each of the three proteins in the immunoprecipitated complex by Western slot blotting. The activity of ACN, but not MDH or ICDH, was enhanced after incubation with calcineurin indicating one possible regulatory function for the complex. The mitochondrial forms of Mn‐SOD, ACN, MDH, and ICDH were identified as partner proteins of calcineurin with all the proteins present in a single multiprotein complex. Proteins 2006. © 2006 Wiley‐Liss, Inc.