Eukaryotic translation elongation factor 1alpha is known to interact in GTP-bound form with aminoacyl-tRNA promoting its binding to the ribosome. In this paper another ternary complex [EF-1alpha*GDP*deacylated tRNA], never considered in widely accepted elongation schemes, is reported for the first time. The formation of this unusual complex, postulated earlier (FEBS Lett. (1996) 382, 18-20), has been detected by four independent methods. [EF-1alpha*GDP]-interacting sites are located in the acceptor stem, TpsiC stem and TpsiC loop of tRNA(Phe) and tRNA(Leu) molecules. Both tRNA and EF-1alpha are found to undergo certain conformational changes during their interaction. The ability of EF-1alpha to form a complex with deacylated tRNA indicates that the factor may perform an important role in tRNA and aminoacyl-tRNA channeling in higher eukaryotic cells.