Histidinol dehydrogenase (HDH), a Zn-metalloenzyme, produces His from histidinol through two successive oxidation reactions with NAD+ as a coenzyme. A mutation, His261-->Asn, caused the complete loss of the Zn, thereby inactivating the enzyme, without significant structural perturbation. The ability to oxidize an intermediate, histidinaldehyde, was restored to about 4% of that of the wild-type enzyme by adding 0.5 mM MnCl2, whereas the histidinol oxidation activity could not be recovered with the mental addition. We concluded that the His residue at position 261 is essential for the ligation of the Zn of cabbage HDH.