Project Abstract In the United States (U.S.), demand for red blood cells (RBCs) for use in transfusion medicine is steadily growing and will exceed the supply with a projected shortage of 4 million units of RBCs by the year 2030. In addition, frequent seasonal blood shortages, decreasing donation rates, risks of disease transmission, the RBC storage lesion and other side-effects threaten the existing supply of RBCs. Therefore, a viable short-term alternative to donated human RBCs must be developed which is safe, available in large quantities at low cost, and free of the risks of disease transmission and immune suppression, as well as address concerns over religious objections to receiving transfused blood. RBC substitutes should be used to treat conditions in which the use of banked blood is unreasonable or for which there is no therapy. Despite decades of research and clinical trials, mammalian hemoglobin (Hb)-based oxygen (O2) carriers (HBOCs) are still plagued by severe side-effects, such as vasoconstriction at the microcirculatory level, systemic hypertension, myocardial infarction and increased mortality rates. It has been hypothesized that the side-effects associated with previous generations of HBOCs were caused by HBOC extravasation through the blood vessel wall into the tissue space, which led to scavenging of nitric oxide (NO). NO is also known as endothelium-derived relaxing factor (EDRF), and the endothelium (inner lining) of blood vessels uses NO to signal the surrounding smooth muscle to relax, resulting in vasodilation and increased blood flow. Previous generations of HBOCs were also easily oxidized in vivo, creating a significant amount of oxidative stress and tissue damage. All of these problems are a direct consequence of removing Hb from the protective environment of the RBC. We propose that many of these side-effects may be prevented or reduced by using naturally occurring acellular Earthworm Hb (erythrocruorin, LtEc), which has evolved to exist outside of RBCs. Unlike other Hbs (64 kDa), LtEc is naturally large (3.6 MDa) and should not extravasate through the pores lining the blood vessel wall. This feature should prevent/limit its vasoactivity and the extent of systemic hypertension. LtEc is also extremely stable and resistant to oxidation. Therefore, it should elicit limited oxidative stress and tissue toxicity compared to acellular HBOCs synthesized from mammalian Hbs. In addition, LtEc appears to have a much slower or non-existent NO scavenging rate compared to mammalian Hbs, which is the hypothesized root cause of vasoconstriction and hypertension observed in previous generations of acellular HBOCs. Interestingly, the O2 transport characteristics of LtEc are similar to human RBCs, which suggests it will effectively transport O2 to tissues and organs in vivo. Polyethylene glycol (PEG) conjugation to the surface of purified LtEc camouflages the molecule against recognition by the immune system and increases LtEc circula...