Initiation of polypeptide synthesis in a cell-free system of Escherichia coli directed by alfalfa mosaic virus RNA 4 was studied by using either fMet-tRNA or Ac-Phe-tRNA as initiator tRNA. Initiation with fMet-tRNA yielded a product that was identical to the authentic viral coat protein except that the NH2-terminal serine was preceded by fMet instead of being acetylated. When Ac-Phe-tRNA was used as initiator, the biosynthetic product was 10-12 amino acid residues longer, the extra amino acids being located at the NH2-terminus. fMet-tRNA and Ac-Phe-tRNA did not compete for ribosomes during initiation of protein synthesis, as became evident from incorporation studies using both initiator tRNAs simultaneously. It is concluded that E. coli ribosomes recognize two sites on the 5' end of alfalfa mosaic virus RNA 4 that are separated by a region of about 30 nucleotides. The results are in complete agreement with the 5'-terminal nucleotide sequence of this RNA [Koper-Zwarthoff, E. C., Lockhard, R. E., RajBhandary, U. L., Alzner-deWeerd, B. & Bol, J. F. (1977) Proc. Natl. Acad. Sci. USA 74, 5504-5508].