The aim of this study was to investigate whether reoxygenation with 21% O2 rather than 100% O2 results in reduced hydrogen peroxide (H2O2) concentrations in neutrophils (PMN). Piglets (2-4 d old) exposed to severe hypoxia (inspired fraction of oxygen, 0.08) were randomized to resuscitation with 21 (n = 13) or 100% O2 (n = 12). Five animals served as controls. H2O2 concentrations in PMN in terms of rhodamine 123 (Rho 123) fluorescence intensity from arterial and superior sagittal sinus blood were quantified by flow cytometry. Laser Doppler flowmetry (LDF) was used to assess cortical blood perfusion. During hypoxia, Rho 123 increased in arterial PMN in both study groups by 15 and 32%, respectively (p < 0.05). In cerebral venous PMN, the increase was less dominant (p = 0.06). Reoxygenation with 100 or 21% O2 had no different effect on Rho 123 in arterial PMN. In cerebral venous PMN, Rho 123 was approximately 40% higher after 60 min and 30% higher after 120 min compared with corresponding data in the 21% O2 group (p < 0.05), which were close to baseline levels. Further, O2 treatment in both groups induced PMN accumulation in arterial blood (p < 0.05). Laser Doppler flowmetry signals increased during transient hypoxia (p < 0.0001 compared with baseline) and were normalized after reoxygenation in both study groups. In conclusion, arterial and cerebral venous H2O2 concentration in PMN tended to increase during hypoxia. During reoxygenation, H2O2 concentration in PMN in the cerebral circulation was low with 21% O2 but remained high with 100% O2 ventilation. We speculate that oxygen should be reintroduced with more caution during neonatal resuscitation.