Transfer-messenger RNA (tmRNA) is a stable RNA in bacteria of 360 ± 40 nucleotides that can be charged with alanine and can function as both tRNA and mRNA. Ribosomes that are stalled either in a coding region of mRNA or at the 3′ end of an mRNA fragment lacking a stop codon are rescued by replacing their mRNA for tmRNA. Here we demonstrate that the interaction of tmRNA with the elongation factor Tu shows unexpected features. Deacylated tmRNA can form a complex with either EF-Tu·GDP or EF-Tu·GTP, the association constants are about one order of magnitude smaller than that of an Ala-tRNA·EF-Tu·GTP complex. tmRNA as well as Ala-tmRNA can be efficiently cross-linked with EF-Tu·GDP using a zero-length cross-link. The efficiency of cross-linking in the case of deacylated tmRNA does not depend on an intact CCA-3′ end and is about the same, regardless whether protein mixtures such as the post-ribosomal supernatant (S100 enzymes) or purified EF-Tu are present. Two cross-linking sites with EF-Tu·GDP have been identified that are located outside the tRNA part of tmRNA, indicating an unusual interaction of tmRNA with EF-Tu·GDP.