Histidine-21 is the sole histidine present in the A chain of diphtheria toxin and recent evidence suggests that it is involved in NAD+ binding. Fluorimetric assays of NAD+ binding and diethylpyrocarbonate modification performed at different pH values provide further insights on the role of this residue and indicate that its pKa value is 6.3. Conformational changes of subunit A of diphtheria toxin have been detected by analysis of tryptophan fluorescence in the pH 2.5-4 and pH 9-10.5 ranges. This indicates that histidine-21 is unlikely to be involved in the low pH-driven conformational change of diphtheria toxin.