The safety and efficacy of a conjugate of pyridoxalated hemoglobin and polyethylene glycol (pyridoxalated PEG hemoglobin) were evaluated after administration to rats. The LD50 (lethal dose for 50% survival of group) of pyridoxalated polyethylene glycol (PEG) hemoglobin was greater than 200 ml/kg. Any pro- or anticoagulation activity was not demonstrated in in vitro coagulation tests. One day after 70% exchange-transfusion with pyridoxalated PEG hemoglobin, slight elevations of the serum glutamic-oxaloacetic transaminase, serum glutamic-pyruvic transaminase, and blood urea nitrogen values, which were 101.7 +/- 22.6 IU/L, 33.3 +/- 7.2 IU/L, and 23.1 +/- 1.4 mg/dl, respectively, were observed. However, these values were in the normal range after 3 days. With greater than 90% exchange-transfusion, all rats exchange-transfused with pyridoxalated PEG hemoglobin survived for greater than 2 weeks in contrast to the death of all the rats exchange-transfused with stroma-free hemoglobin or albumin.