In order to elucidate the functional role of single amino acid residue, histidine or tyrosine of neurophysin I for the binding ability of the protein, these amino acids have been modified photochemically or chemically and their effects on the binding abilities for oxytocin and [8-arginine] vasopressin have been investigated. Upon irradiation of NP-I with visible light in the presence of rose bengal and oxygen, single histidine residue was photooxidized very rapidly without any decrease in the hormones-binding ability. On the other hand, single tyrosine residue was found to be photooxidized almost completely after 240 min of irradiation with decrease in the hormone-binding ability. Neither significant changes in other amino acid residues nor peptide bond rupture were found even after 240 min of irradiation. The decrease in the binding ability of the photooxidized protein proceeds almost identically for oxytocin and vasopressin as the ligand. O-Acetylation of the tyrosine residue of NP-I with N-acetylimidazole gives no significant effects on the hormone-binding ability. These findings suggest that the single histidine residue of NP-I has no contribution to the binding process, while the single tyrosine residue, particularly its aromatic ring, of NP-I may participate with the binding process of the protein to both oxytocin and vasopressin with similar contribution.