['In this study we investigated whether storage of red blood cells (RBCs) leads to alterations in nitrite reductase activity, hence in altered hypoxia-induced nitric oxide (NO) bioavailability and methemoglobin formation.', 'Hypoxia-induced NO bioavailability and methemoglobin formation were measured in vitro after nitrite administration to fresh (<1 week of storage) and aged (5-6 weeks of storage) human RBC units and in blood samples of hemodiluted rats subjected to hypoxic ventilation after transfusion with fresh or aged human RBCs.', 'In vitro, NO and methemoglobin levels 10 minutes after nitrite administration were lower in the fresh RBC samples compared to the aged RBC samples (p\u2009=\u20090.026 and p\u2009=\u20090.022, respectively). In vivo, NO bioavailability was also significantly lower in the rats receiving fresh RBCs compared to the group receiving aged RBCs (p\u2009=\u20090.003). In line with NO bioavailability, methemoglobin levels were higher, albeit not significantly, in the group receiving aged RBCs compared to in the group receiving fresh RBCs (p\u2009=\u20090.154). The difference in methemoglobin formation after nitrite administration between fresh and aged RBCs was only present under deoxygenated conditions and not under oxygenated conditions. There were no differences in methemoglobin reductase activity between fresh and aged RBCs.'] Does red blood cell storage increase hypoxia-induced nitric oxide bioavailability and methemoglobin formation in vitro and in vivo?