The amino acid sequence of elongation factor Tu (EF-Tu) from Escherichia coli has been determined. EF-Tu is a single-chain polypeptide composed of 393 amino acids (Mr 43,225 for the species bearing COOH-terminal serine). The NH2-terminal serine is acetylated, and lysine-56 is partially methylated. The sites of facile tryptic cleavage are at arginines 44 and 58 and at lysine-263. The cysteinyl residues associated with aminoacyl-tRNA and guanosine nucleotide binding activities are residues 81 and 137, respectively. The COOH-terminal amino acid is heterogenous in that analyses of the COOH-terminal peptides isolated from different EF-Tu preparations gave position 393 as glycine and serine in ratios (Gly/Ser) ranging from about 0.7 to 3.