In this study endotoxin was removed from hemoglobin preparations using an affinity chromatographic column. The effects of ionic strength and pH on endotoxin removal efficiency and protein recovery have been investigated. Both ionic strength and pH had significant effects on endotoxin removal from hemoglobin solutions. When the ionic strength was increased from 0 to 0.25 M, endotoxin removal efficiency was reduced from 71 to 38% (samples prepared with NaCl solutions), and from 71 to 37% (samples prepared with CaCl2 solutions). When pH was varied from 4.5 to 9.0, endotoxin removal efficiency was reduced from 92 to 43%. However, both ionic strength and pH had very limited effects on protein recovery in the process for hemoglobin-LPS feeds as well as pure hemoglobin feeds. Hemoglobin recovery was above 94% in all cases.