The protonation states of buried histidine residues in human deoxyhemoglobin were unambiguously identified by using a neutron crystallographic technique. Unexpectedly, the neutron structure reveals that both the alpha- and beta-distal histidines (His alpha 58 and His beta 63) adopt a positively charged, fully (doubly) protonated form, suggesting their contribution to the Bohr effect. In addition, the neutron data provide an accurate picture of the alpha(1)beta(1) hydrogen-bonding network and allow us to observe unambiguously the nature of the intradimeric interactions at an atomic level.