The requirement for GTP in the initiation process on reticulocyte ribosomes and ribosomal subunits has been examined by studying Met-tRNA(F) binding, ribosome-dependent [gamma-(32)P]GTP hydrolysis, and peptide-bond formation with puromycin. Met-tRNA(F) binding can be obtained with the methylene analogue, 5'-guanylylmethylene diphosphonate, as well as GTP, and it is not inhibited by fusidic acid or several other inhibitors of protein synthesis. This reaction can be performed with the 40S subunit and has the same requirements as the Met-tRNA(F)-binding reaction with washed ribosomes. Ribosome-dependent [gamma-(32)P]GTP hydrolysis can be obtained with the initiation factor M(2A) using either washed ribosomes or the 40S subunit. This reaction is also not significantly inhibited by fusidic acid. Peptide-bond formation between puromycin and Met-tRNA(F), however, is inhibited by fusidic acid, and does not occur if the methylene analogue of GTP is substituted for GTP. These data suggest that the binding of the initiator tRNA to the 40S subunit does not require the hydrolysis of GTP, but that at least one GTP hydrolysis event must occur after Met-tRNA(F) binding in order for the first peptide bond to be formed.