Histidinol dehydrogenase (EC 1.1.1.23) from Salmonella typhimurium is inhibited by formaldehyde and pyridoxal 5-phosphate (pyridoxal-P). epsilon-Pyridoxyl-lysine is isolated upon acid hydrolysis of pyridoxal-P-treated enzyme reduced by sodium borohydride. In the presence of formylhistidinol and formylhistidine (specific ligands of the enzyme) inactivation of histidinol dehydrogenase by pyridoxal-P is prevented. Extrapolation of the initial part of the inactivation curve caused by pyridoxal-P indicates that modification of two essential lysine residues results in inactivation of the dimeric enzyme. The essential lysine residues appear to participate in the reversible oxidation/reduction reaction converting histidinol to histidinal.