The Escherichia coli (Es. coli) protein Fis (factor for inversion stimulation) stimulates site-specific DNA inversion of the G segment in phage Mu by binding to a recombinational enhancer. By using synthetic oligonucleotides deduced from the amino-terminal amino acid sequence, we have cloned the gene (termed fis) encoding this specific DNA-binding protein. The DNA sequence shows that the Fis protein is basic and contains 98 amino acids. A helix-turn-helix sequence motif characteristic of many DNA-binding proteins is located at the carboxyl-terminal end of the protein. By marker exchange, we have constructed an insertion mutation of fis. Fis is nonessential for Es. coli growth; however, inversion of the G segment of a Mu prophage was not detected in the fis mutant. The fis gene is located between 71 and 72 min on the Es. coli genetic map.