We aimed to study the influence of preventing methemoglobin (metHb) formation, in the roles of peroxiredoxin 2 (Prx2), glutathione peroxidase (GPx) and catalase (CAT) on the erythrocyte antioxidant defense system. We performed in vitro assays using healthy erythrocytes, with and without inhibition of autoxidation of Hb (saturation with carbon monoxide), followed by H 2 O 2 -induced oxidative stress. We assessed the enzyme activities and amounts of CAT, GPx and Prx2 in the red blood cell (RBC) cytosol and membrane and several biomarkers of oxidative stress, such as the reduced and oxidized glutathione levels, thiobarbituric acid reactive substances (TBARS) levels, membrane bound hemoglobin and total antioxidant status. When autoxidation of Hb was inhibited, no significant changes were found for GPx and CAT; Prx2 was observed only in the monomeric form in the cytosol and none bound to the membrane. Blocking the function of Hb as a pseudo-peroxidase does not seem to have an impact on the function of the RBC peroxidases.