Subunits of rat liver ribosomes have been prepared. Polyphenylalanine is synthesized when both subunits are incubated with [14C]phenylalanyl-tRNA, GTP and transferases I (aminoacyl-tRNA-binding factor) and II (translocation factor); individually, the subunits are inactive in polypeptide biosynthesis. When the small subunit is incubated with transferase I, the particle sedimented from the reaction mixture contains bound transfer factor, and can carry out protein synthesis from [14C]phenylalanyl-tRNA in the absence of transferase I, when supplemented with the large subunit, GTP, and transferase II. The transferase I-small subunit interaction requires aminoacyl-tRNA and polyuridylate, but not GTP; the large subunit does not interact with transferase I and it inhibits partly the reaction with the small subunit. Similar experiments with transferase II reveal that binding of this transfer factor occurs only when both subunits are present. This interaction requires GTP and is stimulated by polyuridylate. © 1969.