tmRNA facilitates a novel translation, trans-translation, in which a ribosome can switch between translation of a truncated mRNA and the tmRNA's tag sequence. The mechanism underlying resumption of translation at a definite position is not known. In the present study, the effects of mutations around the initiation point of the tag-encoding sequence of Escherichia coli tmRNA on the efficiency and the frame of tag translation were assessed by measuring the incorporations of several amino acids into in vitro poly (U)-dependent tag-peptide synthesis. One-nucleotide insertions within the tag-encoding region did not shift the frame of tag translation. Any 1-nt deletion within the span of −5 to −1, but not at −6, made the frame of tag translation heterologous. Positions at which a single base substitution caused a decrease of trans-translation efficiency were concentrated within the span of −4 to −2. In particular, an A-4 to C-4 mutation seriously damaged the trans-translation, although this mutant retained normal aminoacylation and ribosome-binding abilities. A possible stem and loop structure around this region was not required for trans-translation. It was concluded that the tag translation requires the primary sequence encompassing −6 to +1, in which the central 3 nt, A-4, G-3, and U-2, play an essential role. It was also found that several base substitutions within the span of −6 to −1 extensively shifted the tag-initiation point by −1.