An epitope of elongation factor Tu (EF-Tu), which is found in organisms in both the bacterial and archaeal domains, was recently defined by mAb 900. To localize the conserved epitope within the EF-Tu molecule and to determine its sequence, SPOTScan analysis of synthetic peptides, Western blot analysis of purified EF-Tu domains and site-directed mutagenesis studies were used. Analysis of mAb 900 binding to overlapping 15-mer peptides encompassing the complete sequence of EF-Tu of Escherichia coli was inconclusive, suggesting three distinct regions may be epitopes. Western blot analysis of EF-Tu domains 1-3 of Thermus thermophilus suggested that the epitope was located at the N terminus. This was confirmed by site-directed mutagenesis of EF-Tu domain 1 of Mycoplasma hominis. By C-terminal truncation of the N-terminal 15-mer peptide the epitope was mapped to EF-Tu residues 1-6. Replacement of each of the residues in the epitope peptide demonstrated that only positions 5 and 6 were indispensable for antibody binding. These data provide evidence that the highly conserved epitope recognized by mAb 900 in the bacterial and archaeal domains is located at the very end of the N terminus of the EF-Tu molecule.