Abstract Ribosome recycling factor, referred to as RRF, is essential for bacterial growth because of its activity of decomposition of the post-termination complex of the ribosome after release of polypeptides. In this study, we isolated a conditionally lethal amber mutation, named frr-3, in the Escherichia coli RRF gene at amino acid position 161, showing that the truncation of the C-terminal 25 amino acids of RRF is lethal to E. coli. An RRF gene cloned from Thermus thermophilus, whose protein is 44% identical and 68% similar to E. coli RRF, failed to complement the frr-3(Am) allele. However, truncation of the C-terminal five amino acids conferred intergeneric complementation activity on T. thermophilus RRF, demonstrating the modulator activity of the C-terminal tail. Rapid purification of T. thermophilus RRF was achieved by T7-RNA polymerase-driven overexpression for crystallography.