Pyrimidine 5′-nucleotidase (P5′N) partial deficiency has been described in several hematological disorders and also in the β-thalassemic trait. To check if the P5’N deficiency in thalassemia was acquired we used thalassemic red cells (from either homo- or heterozygous subjects), whose P5′N activity was lower than in control cells. After separation on a density gradient, activity in lighter cells was similar to controls and less than 50% in denser cells. The most probable mechanism for this faster inactivation involves enzyme -SH groups modification by oxidation and reaction with monofunctional aldehydes produced by membrane lipid peroxidation. In vitro challenge of thiol enzymes as pyruvate kinase (PK), adenylate kinase (AK) and P5′N with increasing concentrations of GSSG, hexanal (HEX) and 4-hydroxynonenal (HNE), showed that HNE is the most powerful among the enzyme inhibitors tested and that P5′N activity is a more sensitive index of -SH groups damage, when compared to PK and AK.