AbstractPartially purified preparations of two protein factors, one of which is stable at 50°C (FIs) and the other unstable at 50°C (FIu), are required for the GTP‐dependent, mRNA‐directed binding of aminoacyl‐tRNA to ribosomes, as well as for polypeptide synthesis in the presence of a third factor, FII. Both FIsand FIuare required for maximal interaction with GTP to form a GTP•protein complex that subsequently interacts with aminoacyl‐tRNA, but not with deacylated tRNA or withN‐(substituted)‐aminoacyl‐tRNA, to form an aminoacyl‐tRNA•GTP•protein complex. A mixture of FIsand FIualso interacts with GDP to form a GDP•protein complex; however, no subsequent interaction with aminoacyl‐tRNA is observed. In addition to aminoacyl‐tRNA and GTP, Mg2+and NH4+are required for the formation of the aminoacyl‐tRNA•GTP•protein complex. Although both protein factors, FIsand FIu, are required for the formation of this complex, only the heat‐labile protein, FIu, is a component of the complex. Very little dissociation of the GTP moiety of the complex occurs in the presence of Mg2+, and no detectable exchange is observed with GTP, GDP, or Pi. In contrast, appreciable dissociation of the aminoacyl‐tRNA from the GTP•protein occurs even in the presence of Mg2+, and exchange with other aminoacyl‐tRNA's can be readily demonstrated. In the absence of Mg2+, complete dissociation of both the GTP and the aminoacyl‐tRNA from the protein occurs. Evidence has been obtained to demonstrate that the aminoacyl‐tRNA•GTP•protein complex is an intermediate in the GTP‐dependent binding of aminoacyl‐tRNA to ribosomes. The binding of the aminoacyl‐tRNA to the ribosome occurs with the concomitant formation of Piand a GDP • protein complex. Incorporation of the bound aminoacyl‐tRNA into polypepti