Neuroglobin is a recently discovered member of the globin superfamily which is suggested to enhance the O2 supply of the vertebrate brain. Spectral measurements with human and mouse recombinant neuroglobin provide evidence for a hexacoordinated deoxy ferrous (Fe2+) form, indicating a His-Fe2+-His binding scheme. O2 or CO can displace the endogenous protein ligand, which is identified as the distal histidine by mutagenesis. The ferric (Fe3+) form of neuroglobin is also hexacoordinated with the protein ligand E7-His and does not exhibit pH dependence. Flash photolysis studies show a high recombination (kon) and a slow dissociation rate (koff) for both O2 and CO, indicating a high intrinsic affinity for these ligands. However, as the rate limiting step in ligand combination with the deoxy hexacoordinated form involves the dissociation of the protein ligand, O2 and CO binding is suggested to be slow in vivo. Due to this competition, the observed O2 affinity of recombinant human neuroglobin is average (1 Torr at 37°C). Neuroglobin has a high autoxidation rate, resulting in an oxidation at 37°C by air within a few minutes. The oxidation/reduction potential of mouse neuroglobin, E’o = -129 mV, lies within the physiological range. Under natural conditions, recombinant mouse neuroglobin occurs as a monomer with disulfide dependent formation of dimers. The biochemical and kinetic characteristics are discussed in view of the possible functions of neuroglobin in the vertebrate brain.