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Two alternative procedures for the isolation of the elongation factor Tu from Thermus thermophilus were compared and the properties of a specifically nicked EF-Tu . GDP were examined in detail. Although the native elongation factor possessed similar catalytic activities in all reactions investigated as the protein isolated by Arai et al. [Eur. J. Biochem. 92, 509-519 and 521-531 (1978)] it could not be crystallized. The nicked EF-Tu, consisting of two associated fragments with molecular weights of 41000 and 8000 respectively, was active in binding GDP, GTP and in the formation of Phe-tRNAPhe . EF-Tu . GTP ternary complex. However, it did not promote poly(U)-dependent synthesis of polyphenylalanine on Escherichia coli ribosomes. The isolated fragment of a molecular weight of about 41000 did not bind GDP. This activity could be reconstituted with the supplement of the small 8000-Mr fragment. It is demonstrated that, in contrast to the native EF-Tu, the nicked EF-Tu forms high-molecular-weight aggregates. Cleavage of the polypeptide chain of EF-Tu from T. thermophilus stimulates the crystallization of this protein.