Bacillus subtilis RecU protein is involved in homologous recombination, DNA repair, and chromosome segregation. Purified RecU binds preferentially to three- and four-strand junctions when compared to single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) (≈10- and ≈40-fold lower efficiency, respectively). RecU cleaves mobile four-way junctions but fails to cleave a linear dsDNA with a putative cognate site, a finding consistent with a similar genetic defect observed for genes classified within the ε epistatic group (namely ruv A, rec D, and rec U). In the presence of Mg ²⁺ , RecU also anneals a circular ssDNA and a homologous linear dsDNA with a ssDNA tail and a linear ssDNA and a homologous supercoiled dsDNA substrate. These results suggest that RecU, which cleaves recombination intermediates with high specificity, might also help in their assembly.