The purpose of the present study was to investigate the pharmacokinetics of PEG-hemoglobin SB1, a modified bovine hemoglobin with polyethylene glycol, after its single and multiple administration in beagle dogs. For this purpose, the analytical method of free hemoglobin in the plasma was developed and validated. Excellent linearity (r2=0.999) was observed in the calibration curve data, with the limit of quantification of 0.005 g/dL. The precision and the deviation of the theoretical values for accuracy were always within +/-15% in both the between- and the within-day results. The method was tested by measuring the plasma concentrations following intravenous administration to beagle dogs and was shown to be suitable for pharmacokinetic studies. In a single dose study, the plasma half-life (t1/2) increased and the total body clearance (CLt) decreased with the dose (i.e., 0.017 to 0.75 gHb/kg as PEG-hemoglobin SB1) in both sexes. The volume of distribution at steady-state (Vd(ss)) showed no difference with the dose. In contrast, the values of t1/2, CLt and the area under the plasma concentration-time curve (AUC) after the multiple dose were significantly different from those of the single dose administration. The values of t1/2 in the multiple administration were about two times higher than that of the single dose. As a result, t1/2 of hemoglobin after the administration of PEG-hemoglobin SB1 was about 15-30 h, indicating the PEG modification of the hemoglobin lead to a prolongation of plasma concentration of the protein. Therefore, these observations suggested that the PEG modification of hemoglobin is potentially applicable in the hemoglobin-based therapeutics.