Nerve cells are highly susceptible to ischemic and hypoxic injuries. The neuroglobin (Ngb), found in vertebrate nerve cells, has been suggested to protect nerve cells from ischemic episodes by a yet unknown mechanism. However, contradicting reports exist regarding localization and up‐regulation of Ngb in response to hypoxia. The aim of the present study was to probe the distribution of Ngb proteins in mouse brain and retina by immunohistochemistry, and to quantify the levels of Ngb mRNA by reverse‐transcription‐polymerase chain reaction (RT‐PCR) after short‐term (2 h) exposure to 7.6% oxygen. We found Ngb to be present throughout the neocortex, most abundantly in the perirhinal, entorhinal and temporal cortical areas, the thalamus and hypothalamus, the choroid plexus, the olfactory bulb and the cranial nerve nuclei in the brainstem. Intense staining was observed in the mesencephalic central grey area and the Purkinje cells. Two‐hour hypoxic exposure caused no detectable changes in staining intensity or spatial distribution of Ngb neither in the Purkinje cells nor in any other brain areas observed. The RT‐PCR data supported the lack of differences in brain Ngb levels between normal and oxygen‐deprived animals. In the retina, Ngb localization by immunohistochemistry was confined to the inner segments of the photoreceptors, the plexiform layers and the ganglion cells. Short‐termed hypoxia did not change retinal Ngb levels as assessed by both techniques. The lack of Ngb up‐regulation in the brain is consistent with results from previous long‐term hypoxic experiments, suggesting that Ngb is not regulated by pure hypoxia in vivo.