Hemoglobin-based O₂ carriers (HBOC) are undergoing extensive development as potential "blood substitutes." A major problem associated with these molecules is an increase in microvascular permeability and peripheral vascular resistance. In this paper, we utilized bovine lung microvascular endothelial cell (BLMVEC) monolayers and simultaneously measured hemoglobin-induced changes in transendothelial electrical resistance (TER), diffusive albumin permeability (P_DA) and diffusive hemoglobin permeability (P_DH) for three forms of hemoglobins (Hb): natural tetrameric human Hb-A, and two polymerized recombinant HBOCs containing alpha human and beta bovine chains designated Hb-Polytaur, (MW 500 kDa) and Hb-(Poytaur)n (approx. MW 1 million Da). Hb-Polytaur and Hb-(Poytaur)n are being evaluated for use clinical use as HBOCs. All three Hb molecules induced a rapid decline of TER to 30% of baseline. PDA increased, on average, approximately 9-fold (2.78 x 10^-7 vs. 2.47 x 10^-6 cm/sec) in response to Hb exposure. All three Hb molecules induced an increase in their own permeability, a process that we have called hemoglobin-induced hemoglobin permeability. The magnitude of change of P_DH was also related to Hb size. When P_DH was corrected for the diffusive coefficient (D) for each hemoglobin species, no evidence of restricted diffusion was found. Immunofluorescent images demonstrate hemoglobin-induced actin stress fiber formation and large intercellular gaps. These data provide the first quantitative assessment of the effect of polymerized HBOC on their own diffusion rates over time. We discuss the importance of these findings in terms of hemoglobin extravasation rates, molecular sieving and clinical consequences of HBOC use.