A previously isolated mutant of E. coli K12, HAK 88 [Kuwano, M., Endo, H. & Yamamoto, M. (1972) J. Bacteriol. 112, 1150-11561, contains a new protein that in two-dimensional gel electropherograms has the same molecular weight as normal elongation factor Tu, but whose isoelectric point is altered approximately 0.1 pH unit in the acidic direction. Peptide mapping, purification proerties, and the ratio of leucyl plus isoleucyl residues to methionyl plus cysteinyl residues of the normal elongation factor Tu protein and the new protein show a close similarity between the two. The mutation causing the altered electrophoretic mobility is located between argH and rif (79 min on the E. coli genetic map). These biochemical and genetic data indicate that strain HAK 88 has a mutationally altered tufB gene. The development of two-dimensional polyacrylamide electrophoresis (1) has made it possible to identify and study individual components of a complex mixture of proteins, such as those in a crude cell extract. In a continuation of our physiological and genetic studies of elements of the transcription and translation apparatus in Escherichia coli, we have analyzed extracts from the previously isolated putative mutant of elongation factor Ts (EF-Ts), HAK 88 (2), on twodimensional gels. Much to our surprise, extracts of this mutant evidently contained two spots for elongation factor Tu (EF-Tu) which differed from each other in isoelectric point by approximately 0.1 pH unit. We report here the evidence for this and show that the mutation leading to the alteration in one of the EF-Tu For partial purification of EF-Tu from HAK 88 the first three purification steps of the procedure of Arai et al. (3) were used. The procedure was scaled down 100-fold; after the 37-64% (NH4)2SO4 precipitation, the precipitate was dissolved in buffer of Arai et al. (3) and diluted to a conductivity equivalent to that of 0.1 M (NH4)2SO4. After application of the cell extract to the DEAE-Sephadex column the column was washed with 0.2 M KCI buffer. Elution was carried out in a stepwise manner with increasing concentrations of KCI, ranging in steps of 0.02 M, from 0.22 M to 0.30 M.Preparation of Tryptic Peptides. Stained spots of each protein containing 150,000-300,000 cpm were cut from the two-dimensional gel of 85SO4-2-labeled HAK 88 extract.Seven hundred micrograms of salt-free unlabeled EF-Tu (E. coli ), which had previously been denatured in 6 M guanidine-HCl, was added to each of the gel slices. The mixture was suspended in 150 ,l of 0.1 M NH4HCO3, and 50 ,g of (L-1-tosylamido-phenylethyl chloromethyl ketone)-treated trypsin was added (4).