A factor present in rat liver supernatant catalyzes binding of Phe-tRNA to 40S ribosomal subunits from rat skeletal muscle. This factor could be distinguished from aminoacyltransferase I by a number of criteria: (1) at lower concentrations of magnesium (5 mM) the 40S binding factor was approximately seven times as effective as T-I in catalyzing binding of Phe-tRNA to 40S subunits; (2) the kinetics of the binding reaction were different when catalyzed by the 40S binding factor, in particular the initial rate was greater than in the presence of T-I-indeed, the kinetics of the T-I catalyzed reaction resembled nonenzymic binding; (3) GTP was required for maximal binding of Phe-tRNA to 40S subunits in the presence of the 40S binding factor, but not for the T-I catalyzed reaction; (4) the 40S binding factor was inactivated by N-ethylmaleimide whereas T-I was not; (5) finally, the 40S binding factor was more susceptible to heat inactivation. Binding of aminoacyl-tRNA to 40S ribosomal subunits may be a paradigm for the initiation of protein synthesis, and the 40S binding factor may play a role in the process.