Nitric oxide operates as a mediator of important cell signaling pathways. To explore thiol-based protein modifications in a situation of nitrosative stress, two transgenic mouse models (tg- iNOS+ , tg-iNOS+ /myo-/- ) were used. Protein S-nitrosation revealed no differences between WT and tg-iNOS+ hearts as measured by the biotin-switch assay and 2-D PAGE. Even in the absence of myoglobin (endogenous NO-oxidase) and independent of enhanced NO formation the protein S-nitrosation pattern for nearly all the detected proteins (> 40) remained unchanged in the tg-iNOS+ /myo-/- hearts, with the exception of three proteins. Tandem mass spectrometry uncovered these proteins as peroxiredoxins (Prx II, III, VI). Immunoblotting with specific antibodies revealed upregulation of Prx VI in the tg-iNOS+ /myo-/- hearts. The other proteins found to be S-nitrosated were identified as well. Data mining indicated a significant overlap of these proteins with proteins becoming glutathiolated. Protein glutathiolation was enhanced in the tg-iNOS+ hearts and even more so in the tg-iNOS+ /myo-/- hearts. Protein glutathiolation in our model of nitrosative stress is most likely important to protect protein thiols from irreversible oxidation. The upregulation of antioxidant proteins like Prx VI appears to be an additional mechanism to antagonize an excess of reactive oxygen/nitrogen species. Furthermore, the increased S-nitrosation of the Prxs may serve a new function in the signalling cascade of nitrosative stress.