The amino acid sequence of aspartate aminotransferase from E. coli B was determined by the alignment of seven cyanogen bromide peptides. The established sequence of the subunit was composed of 396 amino acid residues, and the molecular weight was calculated to be 43,573. The sequence was compared with those of the pig cytoplasmic and mitochondrial isoenzymes, showing that nearly 30% of all residues were invariant and that the E. coli enzyme exhibited the same degree of homology (about 40%) with either of them. Although majority of the residues were substituted, the functional residues constituting the active site structure were conserved.