The dif locus (deletion-induced filamentation) of Escherichia coli is a resolvase site, located in the terminus region of the chromosome, that reduces chromosome multimers to monomers. In strains in which this site has been deleted, a fraction of the cells is filamentous, has abnormal nucleoid structure, and exhibits elevated levels of the SOS repair system. We have demonstrated that a 33-bp sequence, which is sufficient for RecA-independent recombination and which shows similarity to the cer site of pColE1, suppresses the Dif phenotype when inserted in the terminus region. Flanking sequences were not required, since suppression occurred in strains in which dif as well as 12 kb or 173 kb of DNA had been deleted. However, location was important, and insertions at a site 118 kb away from the normal site did not suppress the Dif phenotype. These sites were otherwise still functional, and they exhibited wild-type levels of RecA-independent recombination with dif-containing plasmids and recombined with other chromosomal dif sites to cause deletions and inversions. It is proposed that the functions expressed by a dif site depend on chromosome location and structure, and analysis of these functions provides a way to examine the structure of the terminus region.