Ribosome releasing factor (RRF) is responsible for the release of ribosomes from messenger RNA at the termination of Escherichia coli protein biosynthesis (Hirashima, A., and Kaji, A. (1972) Biochemistry 11, 4037-4044). RRF has been partially analyzed by Edman degradation to obtain its amino acid sequence. Based on this analysis, a 47-nucleotide probe was synthesized and used to screen clones from the Clarke and Carbon Gene Bank which carry sequences within the 0-10 min region of the E. coli gene map. The entire RRF cistron was detected on the plasmid pLC6-32. The DNA sequence of RRF was determined, and it was deduced that RRF consists of 185 amino acids with a calculated molecular weight of 20,639. A rho-independent transcriptional termination sequence was found immediately downstream of the RRF cistron. The RRF gene was subcloned into the vector pUC19, and the resulting plasmid was named pRR1. E. coli harboring this plasmid expressed much more RRF than cells containing pUC19, and it was biologically active.