Limited proteolysis of native elongation factor Tu (Mr 44 000) by trypsin occurs in at least three distinct steps. The first intermediate arises through cleavage at a site about 65 residues from the amino-terminal end of the protein. It is functionally active [Jacobson, G. R. & Rosenbusch, J. P. (1976) Biochemistry, 15, 5105-5110] and is partially protected from further degradation by the antibiotic kirromycin. The second step converts this intermediate to one of similar size (Mr 37 000) which now is partially inactivated. It is likely to be identical with the intermediate described by Arai et al. [(1976) J. Biochem. Tokyo, 79, 69-83]. In the third step, the partially inactive intermediate is cleaved without any apparent change in the functional properties tested. The resulting two trypsin-resistant fragments have molecular weights of 24 000 and 14 000, and remain associated under nondenaturing conditions. When either of these polypeptides, after isolation in 8 M urea, is allowed to renature, no significant reactivation of GDP binding is observed unless the isolated fragments are mixed before renaturation. These results show that the two fragments are structurally and functionally interdependent.