Hepatic hollow fiber bioreactors are considered a promising class of bioartificial liver assist device (BLAD). Unfortunately, limited oxygen (O2) transport to hepatocytes within this device hinders further development. Hepatocytes in vivo (in the liver sinusoid) experience a wide range of oxygen tensions (pO2 = 25–70 mmHg), which is important for development of proper differentiated function (zonation). Previously, we observed that bovine red blood cell (bRBC) supplementation of the circulating media stream enhanced oxygenation of cultured C3A hepatoma cells compared to a culture with no O2 carrier (Gordon, J.; Palmer, A. F. Artif. Cells, BloodSubstitutes, Biotechnol.2006, 33 (3), 297–306). Despite this success, the cells were not exposed to the desired in vivo O2 spectrum (Sullivan, J.; Gordon, J.; Palmer, A. Biotechnol. Bioeng.2006, 93 (2) 306–317). We hypothesize that altering the kinetics of O2 binding/release to/from hemoglobin-based O2 carriers (HBOCs) could potentially target O2 delivery to cell cultures. High P50 (low O2 affinity) HBOCs preferentially targeted O2 delivery at high inlet pO2 values. Conversely, low P50 (high O2 affinity) HBOCs targeted O2 delivery at low inlet pO2 values. Additionally, inlet pO2, flow rate, and HBOC concentration were varied to find optimal bioreactor operating conditions. Our results demonstrate that HBOCs can enhance O2 delivery to cultured hepatocytes, while exposing them to in vivo-like O2 tensions, which is critical to create a fully functional BLAD.