Translation of eukaryotic mRNA is initiated by a unique amino-acyl tRNA, Met-tRNAiMet, which passes through a complex series of highly specific interactions with components of the translation apparatus during the initiation process. To facilitate in vitro biochemical and molecular biological analysis of these interactions in fully reconstituted translation initiation reactions, we generated mammalian tRNAiMet by in vitro transcription that lacked all eight base modifications present in native tRNAiMet. Here we report a method for in vitro transcription and aminoacylation of synthetic unmodified initiator tRNAiMet that is active in every stage of the initiation process, including aminoacylation by methionyl-tRNA synthetase, binding of Met-tRNAiMet to eIF2-GTP to form a ternary complex, binding of the ternary complexes to 40S ribosomal subunits to form 43S complexes, binding of the 43S complex to a native capped eukaryotic mRNA, and scanning on its 5′ untranslated region to the correct initiation codon to form a 48S complex, and finally joining with a 60S subunit to assemble an 80S ribosome that is competent to catalyze formation of the first peptide bond using the [35S]methionine residue attached to the acceptor terminus of the tRNAiMet transcript.