The ruvC gene has been cloned into the plasmid pT7-7 under the control of the T7 phi 10 promoter. Following induction with isopropyl-1-thio-beta-D-galactopyranoside, the 19-kDa RuvC protein was overexpressed to 20-30% of total cell protein. RuvC has been purified to homogeneity by a simple procedure involving precipitation from the crude lysate, followed by three chromatographic steps. The purified protein resolves synthetic Holliday junctions (60 nucleotides in length) by cleavage at the 3'-side of a phosphate group, to produce nicked duplex DNA. Under the same conditions no cleavage of linear duplex or single-stranded DNA was detected. However, low levels of cleavage were observed with supercoiled form I and single-stranded circular DNA substrates, consistent with the interaction of RuvC with secondary structures. Using synthetic Holliday junctions, we show that RuvC-mediated resolution requires Mg2+ (10 mM) and exhibits an alkaline pH optimum (pH 9.0). No energy cofactors are needed. When RuvC was analyzed by gel filtration and polyacrylamide gel electrophoresis, monomeric and dimeric forms of the protein were observed.