A special type of transfer ribonucleic acid (tRNA) is employed in the initiation step of protein synthesis. The differences between the tRNAs involved in initiation and elongation establish the specificity and recognition of the tRNAs by the factors and enzymes involved in the initiation and elongation steps of protein synthesis. The important determinants of the initiator tRNA in bacteria are the absence of a Watson–Crick base pair between positions 1 and 72 in the acceptor stem and the presence of three conserved consecutive G:C base pairs in the anticodon stem. Formylation of the methionine attached to this initiator tRNA is an important feature. Conversely, the eukaryotic initiator tRNA is mainly determined by the presence of a particular A1:U72 Watson–Crick base pair in the acceptor stem as well as the nature of the base pairs 50:64 and 51:63 in the TΨC (T) stem. Key concepts Initiator tRNA brings methionine to the initiation complex for initiation of protein synthesis. Structural features of initiator tRNAs ensure that they are recognized by translation initiation factors and discriminated against by translation elongation factors. The important determinants of initiator tRNA in bacteria are the absence of a Watson–Crick base pair between positions 1 and 72 in the acceptor stem and the presence of three conserved consecutive G:C base pairs in the anticodon stem. The important determinants of initiator tRNA in eukaryotes are the presence of a particular A1:U72 Watson–Crick base pair in the acceptor stem as well as the nature of the base pairs 50:64 and 51:63 in the T stem. Bacterial initiator tRNA is aminoacylated by methionyl tRNA synthetase, which mainly interacts with the anticodon. The methionine of bacterial initiator tRNA is formylated by methionyl tRNA transformylase, which mainly recognizes the absence of the 1:72 base pair. Initiation factor IF2 ensures recognition and correct binding of bacterial initiator tRNA to the ribosomal P‐site by interacting primarily with the formyl group and the 3′ end of the acceptor arm. Initiation factor IF3 performs proofreading of the 30S initiation complex by verifying base pairing between 5′C of bacterial initiator tRNA anticodon and 3′G of mRNA initiation codon. Bacterial initiator tRNA undergoes several conformational changes during translation initiation to ensure correct positioning in the P‐site of the ribosome. Bacterial initiator tRNA is not a substrate for peptidyl tRNA hydrolase.