In septic shock, cardiovascular collapse is caused by the release of inflammatory mediators. We previously found that lysozyme (Lzm-S), released from leukocytes, contributed to the myocardial depression and arterial vasodilation that develop in canine models of septic shock. To cause vasodilation, Lzm-S generates hydrogen peroxide (H₂O₂) that activates the smooth- muscle soluble guanylate cyclase (sGC) pathway, although the mechanism of H₂O₂ generation is not known. To cause myocardial depression, Lzm-S binds to the endocardial endothelium resulting in the formation of nitric oxide (NO) and subsequent activation of myocardial sGC, although the initial signaling event is not clear. In this study, we examined whether myocardial depression produced by Lzm-S was also caused by the generation of H₂O₂ and whether Lzm-S could intrinsically generate H₂O₂ as has been described for other protein-types. Methods and Results: In a canine ventricular trabecular preparation, we showed that the peroxidizing agent Aspergillus niger catalase, that would breakdown H₂O₂, prevented Lzm-S- induced decrease in contraction. We also found that Compound I, a species of catalase formed during H₂O₂ metabolism, contributed to the NO generation caused by Lzm-S. In a tissue-free experiment, we used a fluorometric assay (Ultra Amplex Red H₂O₂ assay) and electrochemical sensor techniques, respectively, to measure H₂O₂ generation. We found that Lzm-S could generate H₂O₂ and furthermore that this generation could be attenuated by the singlet oxygen quencher sodium azide. Conclusions: This study shows that Lzm-S, a mediator of sepsis, is able to intrinsically generate H₂O₂. Moreover, this generation may activate H₂O₂- dependent pathways leading to cardiovascular collapse in septic shock.