Native elongation factor G possesses three peptide bonds or limited regions of the molecule which are especially sensitive to trypsinolysis. Cleavage at these sites occurs in sequence. Initially, the protein, a single peptide chain of 74,000 daltons, is rapidly split into a fragment of 71,000 daltons and one or more small peptides totaling 3000 daltons. This initial scission does not alter the gross three-dimentional structure of the protein, as the native and trypsin-cleaved protein have the same Kd on Sephadex gel filtration. Although cleaved, the modified elongation factor G retains full activity as measured by its ability to form complexes with the ribosome and guanine nucleotides. A single peptide bond (or region) in the 71,000-dalton fragment is then completely cleaved, yielding fragments of 47,000 and 29,000 daltons. These fragments do not remain associated under native conditions, as they are clearly resolved on gel filtration. Loss of activity is concomitant with the scission of the 71,000-dalton fragment. The third cleavage is complete under the conditions employed here and occurs in the 47,000-dalton peptide generating a fragment of 45,000 daltons and one or more small peptides totaling 2000 daltons. Prolonged treatment with higher levels of trypsin ultimately reduces the protein to small peptides but without generating further discrete fragments visible on sodium dodecyl sulfate gel electrophoresis.