Mice are increasingly used to study the early molecular mechanisms inducing injury to the heart following myocardial infarction. To date, two-dimensional gel electrophoresis combined with mass spectrometry has not been applied to identify changes in protein expression in myocardial tissue of mice subjected in vivo to permanent ischaemia (PI) or ischaemia-reperfusion (IR). In the PI group, ischaemia was induced for 210 min by ligation of the left anterior descending coronary artery while in the IR group, ischaemia was maintained for 30 min and reperfusion was allowed for 180 min. In both groups, the area of the left ventricle at risk was processed for 2-dimensional gel electrophoresis. By comparing protein density changes in cytosolic as well as membrane fractions, we found a total of 32 protein spots that were differentially expressed. Twenty spots changed in expression level after PI alone, four spots after IR alone, and eight spots changed in both models. Identified proteins with MALDI TOF-TOF and LC-MS/MS can be classified into functional groups of anticoagulant proteins, structural proteins, inflammatory-related proteins, transcription- and translation-related proteins, heat shock proteins (HSPs), metabolism-related proteins and miscellaneous. A remarkable finding was the IR-specific translocation of annexins (A3 and A5) from the cytosolic to the membrane compartment, a phenomenon that was verified by Western blotting. Four proteins were changed in expression level at multiple spot locations, characterized by a difference in isoelectric point. In the case of cardiac troponin T and HSP-20, these changes were also dependent on the model. In addition, one spot for the proteins adenylate kinase 1, cardiac troponin T and HSP-20 was uniquely present in the IR and/or PI groups and not in the respective sham groups. The specific alterations in protein expression that took place after PI and IR may stimulate the search for new tools to diagnoze myocardial infarction and to characterize specific pathology-related changes in protein expression.