Phospholemman (FXYD1, PLM), the principal sarcolemmal substrate for protein kinases A and C in the heart, is a regulator of the cardiac sodium pump. We investigated proteins that interact with PLM in adult rat ventricular myocytes using bifunctional crosslinking reagents and co-immunoprecipitation.Digitonin-permeabilized ventricular myocytes were treated with the heterobifunctional crosslinking reagent sulfo-lc-smpt (distance between reactive groups 2nm, reactive towards amino and sulfhydryl amino acid side chains), and cell lysates immunoblotted for PLM. We found sulfo-lc-smpt quantitatively crosslinked PLM to a 20-25kDa protein (electrophoretic mobility of PLM 15kDa, electrophoretic mobility of crosslinked adduct 37kDa). Co-immunoprecipitation experiments indicated that the crosslinked adduct was PLM linked to the anti-oxidant protein peroxiredoxin 6 (prdx6). PLM phosphorylation at serine 68 (protein kinase A activation with 10μM forskolin) or at serines 63, 68 and threonine 69 (protein kinase C activation with 300nM PMA) had no effect on the ability of sulfo-lc-smpt to crosslink PLM to prdx6. Hydrogen peroxide treatment of ventricular myocytes (1-100μM) was also without effect on the binding of prdx6 to PLM.In conclusion, our data suggest a new role for PLM in the heart. As well as being responsible for kinase-mediated regulation of the cardiac sodium pump, it is responsible for recruitment of prdx6 to this ion transporter. Prdx6 catalyzes the reduction of hydrogen peroxide, fatty acid hydroperoxides and phospholipid hydroperoxides using glutathione. Given the well-established sensitivity of the alpha subunit of the sodium pump to cysteine oxidation, recruitment of prdx6 to the sodium pump by PLM may be important to maintain pump activity during periods of oxidative stress.