In mammalian cells valyl-tRNA synthetase (ValRS) forms a high Mr complex with the four subunits of elongation factor EF-1H. The beta, gamma, and delta subunits, that contribute the guanine nucleotide exchange activity of EF-1H, are tightly associated with the NH2-terminal polypeptide extension of valyl-tRNA synthetase. In this study, we have examined the possibility that the functioning of the companion enzyme EF-1alpha could regulate valyl-tRNA synthetase activity. We show here that the addition of EF-1alpha and GTP in excess in the aminoacylation mixture is accompanied by a 2-fold stimulation of valyl-tRNAVal synthesis catalyzed by the valyl-tRNA synthetase component of the ValRS.EF-1H complex. This effect is not observed in the presence of EF-1alpha and GDP or EF-Tu.GTP and requires association of valyl-tRNA synthetase within the ValRS.EF-1H complex. Since valyl-tRNA synthetase and elongation factor EF-1alpha catalyze two consecutive steps of the in vivo tRNA cycle, aminoacylation and formation of the ternary complex EF-1alpha.GTP. Val-tRNAVal that serves as a vector of tRNA from the synthetase to the ribosome, the data suggest a coordinate regulation of these two successive reactions. The EF-1alpha.GTP-dependent stimulation of valyl-tRNA synthetase activity provides further evidence for tRNA channeling during protein synthesis in mammalian cells.