The blood storage lesion refers to changes in red blood cells (RBCs) during storage. It includes a number of chemical and morphological changes in RBCs, which result in reduced integrity of the erythrocyte membrane with formation of microparticles, and increased cell-free hemoglobin in plasma. The geometry of the red blood cell tends to become more spherical, the mean cell hemoglobin concentration (MCHC) in RBCs decreases, RBC volume varies, and the structure of RBC membrane changes significantly. The adverse effects associated with the blood storage lesion are under investigation. We hypothesize that increased nitric oxide (NO) scavenging due to red cell breakdown contributes to the blood storage lesion. In this study, we examined the rate of cell-free hemoglobin and microparticles reacting with NO and found that microparticles scavenge NO only 3 times slower than cell-free hemoglobin but still about 1000 times faster than RBCs. Thus, release of cell-free hemoglobin and microparticles during storage and post transfusion reduces NO bioavailability. We further explored factors that would determine the extent to which red cell microparticles contribute to NO scavenging, such as the ability of these microparticles to concentrate in the cell free zone. We found that microparticles, like cell-free hemoglobin, enter the cell-free zone and as little as 5 µM hemoglobin encapsulated in microparticles has the potential to reduce NO bioavailability and impair endothelial-dependent vasodilation. Additionally, we examined the rate of NO scavenging by fresh and old stored RBCs and found that old stored RBCs scavenge NO about 2 times faster than fresh stored RBCs. In order to understand the mechanisms of increased NO scavenging by older stored RBCs, we simulated NO scavenging by RBCs using 3D single RBC models. Our work shows that the rate of NO scavenging by RBCs increases as RBC MCHC or volume decreases. RBC membrane permeability needs to increase 5 to 70 fold to compensate the effect of geometry and explain our experimental findings. In summary, we have elucidated the extent and mechanisms of reduced NO bioavailability due to red cell breakdown thereby establishing how it contributes to pathological consequences of the storage lesion.