When the polypeptide initiation sequence is interrupted by the peptide antibiotic edeine, the sedimentation coefficient of the native small ribosomal subunit is increased from 43 S to 48 S. Formation of the 48 SN particle is absolutely dependent on the binding of both Met-tRNAf and globin mRNA. These results support the concept that Met-tRNAf binds to the native small ribosomal subunit before mRNA, and that processes involved in mRNA binding to the 43 SN preinitiation complex may be the rate-limiting step of 80 S initiation complex formation.