Peroxiredoxins (Prdx), a family of antioxidant and redox signalling proteins, are plentiful within the heart, although their cardiac functions are poorly understood. These studies were designed to characterise the complex changes that peroxiredoxins undergo within rat myocardium in response oxidant stress. Hydrogen peroxide, a peroxiredoxin substrate, was used as model oxidant pertinent to redox signalling during health and to injury at higher concentrations. Rat hearts were aerobically perfused by the Langendorff method with a broad concentration range of hydrogen peroxide, homogenised and analysed by immunoblotting. Heart extracts were also analysed by size exclusion chromatography under non-denaturing conditions. Changes in disulphide bond formation, non-reversible oxidation of cysteine (hyperoxidation) and subcellular localisation were determined in response to peroxide. Hydrogen peroxide induced an array of changes in the myocardium, including the formation of disulphide bonds that were intermolecular for Prdx1-3 but intramolecular within Prdx5. For Prdx1, Prdx2, and Prdx5 disulphide bond formation can be approximated to an EC50 range of 10-100µM, 1-10µM and 100-1000µM peroxide, respectively. Hydrogen peroxide induced hyperoxidation, not just within monomeric Prdx (by SDS-PAGE) but also within Prdx disulphide dimers and reflects a flexibility within the dimeric unit. Prdx oxidation was also associated with movement from the cytosolic to both the membrane and myofilament enriched fractions. In summary, peroxiredoxins undergo a complex series of redox dependent structural changes in the heart in response to oxidant challenge with its substrate hydrogen peroxide.