In a recent study, ultrahigh molecular weight (M_w) glutaraldehyde‐polymerized bovine hemoglobins (PolybHbs) were synthesized with low O₂ affinity and exhibited no vasoactivity and a slight degree of hypertension in a 10% top‐load model.¹ In this work, we systematically investigated the effect of varying the glutaraldehyde to hemoglobin (G:Hb) molar ratio on the biophysical properties of PolybHb polymerized in either the low or high O₂ affinity state. Our results showed that the M_w of the resulting PolybHbs increased with increasing G:Hb molar ratio. For low O₂ affinity PolybHbs, increasing the G:Hb molar ratio reduced the O₂ affinity and CO association rate constants in comparison to bovine hemoglobin (bHb). In contrast for high O₂ affinity PolybHbs, increasing the G:Hb molar ratio led to increased O₂ affinity and significantly increased the CO association rate constants compared to unmodified bHb and low O₂ affinity PolybHbs. The methemoglobin level and NO dioxygenation rate constants were insensitive to the G:Hb molar ratio. However, all PolybHbs displayed higher viscosities compared to unmodified bHb and whole blood, which also increased with increasing G:Hb molar ratio. In contrast, the colloid osmotic pressure of PolybHbs decreased with increasing G:Hb molar ratio. To preliminarily evaluate the ability of low and high O₂ affinity PolybHbs to potentially oxygenate tissues in vivo, an O₂ transport model was used to simulate O₂ transport in a hepatic hollow fiber (HF) bioreactor. It was observed that low O₂ affinity PolybHbs oxygenated the bioreactor better than high O₂ affinity PolybHbs. This result points to the suitability of low O₂ affinity PolybHbs for use in tissue engineering and transfusion medicine. Taken together, our results show the quantitative effect of varying the oxygen saturation of bHb and G:Hb molar ratio on the biophysical properties of PolybHbs and their ability to oxygenate a hepatic HF bioreactor. We suggest that the information gained from this study can be used to guide the design of the next generation of hemoglobin‐based oxygen carriers (HBOCs) for use in tissue engineering and transfusion medicine applications. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011