Diphtheria toxin has recently been fractionated into two forms, one of which contains tightly but noncovalently bound nucleotide-like material. Here we report identification of the major nucleotide constituent (comprising 80% of the total extractable ultraviolet-absorbing material) as adenylyl-(3',5')-uridine 3'-monophosphate (ApUp). Upon incubation with ApUp, the nucleotide-free form of toxin bound approximately one molar equivalent of the dinucleotide and was converted to a form similar or identical with the native nucleotide-bound form.