Artificial blood substitutes based on glutaraldehyde cross-linked hemoglobin (PolyHb) are currently being developed for use in human subjects needing blood transfusions. Despite the commercial development of PolyHb dispersions, a systematic study of the effect of varying the glutaraldehyde to hemoglobin (G-Hb) molar ratio on the resulting PolyHb physical properties (molecular weight distribution and oxygen binding parameters) has not been conducted to date. The results of this study show that increasing the G-Hb molar ratio elicits a general decrease in the P-50 (partial pressure of oxygen at which Hb is half saturated with oxygen) and cooperativity and a simultaneous increase in the weight averaged molecular weight (M-w) of the PolyHb dispersion and methemoglobin (MetHb) level. Three PolyHb dispersions (20:1, 30:1, and 40:1 G-Hb molar ratios) displayed potential as artificial blood substitutes. The 20:1 PolyHb dispersion resulted in the presence of more intramolecularly cross-linked and non-cross-linked tetramers versus cross-linked species that were larger than a tetramer (similar to75% tetrameric and similar to25% higher-order species), lower MetHb level (8%), and P-50 (20.1 mmHg) similar in magnitude to that of non-cross-linked Hb. The 30:1 PolyHb dispersion consisted of more higher-order species (similar to76%), higher MetHb level (28%), and lower P-50 (13.3 mmHg). The 40:1 PolyHb dispersion resulted in a similar P-50 of 13.0 mmHg and similar MetHb level (30%); however, this PolyHb dispersion only consisted of species larger than a tetramer. The molecular weight distribution of PolyHb dispersions was determined using asymmetric flow field-flow fractionator (AFFF) coupled with multiangle static light scattering (MASLS). This is the first time that AFFF-MASLS has been used to characterize the molecular weight distribution of PolyHb dispersions.