Transglutaminase 2 (TGase 2) is a Ca2+-dependent enzyme responsible for the posttransttranslational modification of proteins by transamidation of specific polypeptide-bound glutamine residues. Elevating the intracellular concentration of Ca2+-ions in human erythrocytes leads to the formation of cytoskeletal and cytoplasmic protein polymers. The Ca2+-dependent TGase 2-dependent cross-linking activity has been proposed for its involvement in erythrocyte aging, by inducing irreversible modification of their cell shape and deformability. Accordingly, we found that high-density ("old") TGase 2(-/-) red blood cells (RBCs) were more resistant to osmotic stress-induced hemolysis than those from wild type mice. In addition, elevating the intracellular concentration of Ca2+ by treatment of total RBCs with ionophore A23187 resulted in enhanced resistance of TGase 2-deficient erythrocytes compared to their normal counterpart. These findings indicate that TGase 2 may have a role in regulating structural flexibility of RBCs, possibly affecting their life span in physiopathological conditions, such as erythrocyte senescence, which are accompanied by increases in intracellular Ca2+ concentration. (C) 2002 Elsevier Science (USA).