It has been demonstrated that low concentrations of highly purified diphtheria toxin specifically inhibit incorporation of labeled amino acids into polypeptides in extracts from HeLa cells and from rabbit reticulocytes. No inhibition of incorporation occurs in the absence of a specific cofactor. This cofactor has been identified as nicotinamide adenine dinucleotide (NAD). It has been shown that it is one of the steps involving transfer of amino acids from soluble ribonucleic acid to the growing polypeptide chain that is affected by the toxin in the presence of NAD.