The amino acid diphthamide is a complex post-translational derivative of histidine that exists in eukaryotic and Archaebacterial elongation factor 2 (EF-2). Diphtheria toxin and Pseudomonas exotoxin A catalyze the transfer of an ADP-ribose residue from NAD to diphthamide, causing the inactivation of EF-2. We have used cytosolic extracts of mutant CHO-K1 cells to study the biosynthesis of diphthamide in vitro. We have identified chromatographically a precursor form of diphthamide that exists in one complementation group of mutant cells and have documented the addition of 3 methyl residues from S-adenosylmethionine to this precursor. We have identified the presence of methyltransferase capable of carrying out this reaction in vitro in cells of 15 diverse eukaryotic species.