tmRNA (transfer messenger RNA) is a unique molecule used by all bacteria to rescue stalled ribosomes and to mark unfinished peptides with a specific degradation signal. tmRNA is recruited by arrested ribosomes in which it facilitates the translational switch from cellular mRNA to the mRNA part of tmRNA. Small protein B (SmpB) is a key partner for the trans-translation activity of tmRNA both in vivo and in vitro. It was shown that SmpB acts at the initiation step of the trans-translation process by facilitating tmRNA aminoacylation and binding to the ribosome. Little is known about the subsequent steps of trans-translation. Here we demonstrated the first example of an investigation of tmRNA-ribosome complexes at different stages of trans-translation. Our results show that the structural element at the position of tmRNA pseudoknot 3 remains intact during the translation of the mRNA module of tmRNA and that it is localized on the surface of the ribosome. At least one SmpB molecule remains bound to a ribosome-tmRNA complex isolated from the cell when translation is blocked at different positions within the mRNA part of tmRNA.