The hip locus of Escherichia coli affects the frequency of persistence to the lethal consequences of selective inhibition of either DNA or peptidoglycan synthesis. Regulation of the hip operon, which consists of a regulatory region and two genes, hipB and hipA, was examined with strains containing a hip-lac transcriptional fusion placed in single copy at the lambda att site. Disruption of the hip locus increased activity from the fusion 16-fold. Repression was restored by supplying HipB in trans. HipB was overexpressed and purified. On the basis of gel filtration and cross-linking studies, HipB is a dimer in solution. Sequence analysis revealed that HipB is a Cro-like DNA-binding protein. The interaction of HipB with the hip regulatory region was examined by gel retardation, DNase I protection, and methylation protection studies. HipB binds with a Kapp (K apparent) of 40 pM to four operator sites with the conserved sequence TATCCN8GGATA (N represents any nucleotide). Binding to the operators is nearly simultaneous and appears to be cooperative. Analysis of the role of HipA in the regulation of the hip operon is complicated by the toxicity of HipA in the absence of HipB. Strains disrupted in hipB but not in hipA could not be recovered. Moreover, hipA-containing plasmids cannot be replicated in strains defective in or lacking hipB. HipA is found exclusively in a tight complex with HipB. Although disruption of hipA slightly increased expression from the hip-lac fusion, in vitro studies suggest that HipA does not bind to the hip regulatory region directly but indirectly via HipB.