The normally hexa coordinate ferrous form of neuroglobin binds CO by replacement of the heme-linked distal histidine residue. We have studied this reaction in detail using stopped flow techniques. The reaction time courses are complex at all the wavelengths studied. Specifically the reaction with CO occurs in two temporally separable phases, each of which shows a hyperbolic dependence of rate on CO concentration, indicating they each arise from histidine replacement by CO. Analysis of the observed rates as a function of the CO concentration, measured in the pH range 6.0-8.0, allows us to determine both the rate of histidine-heme ligand binding and dissociation for each of the two forms of the protein present in solution at each pH value. The pH dependence of the histidine association and dissociation rates is complex, as are the derived equilibrium constants for distal histidine binding. The spectral change associated with each reaction phase is very similar and independent of the CO concentration, showing that the two protein forms responsible for the two observed kinetic processes are not in equilibrium on the time scale of our investigations. Our data suggests that, unlike many other heme proteins, neuroglobin displays complex reactivity with ligands in the ferrous form due to heme rotational disorder, as has previously been reported for the ferric form of the protein.