Eight synthetic Holliday junction (HJ) oligonucleotides containing an immobile or a mobile junction were characterized by gel electrophoresis, ultraviolet absorption and circular dichroism (CD) spectroscopy. Four 24-mer deoxyribonucleotides formed stable immobile and mobile HJs in 0.1 M NaCl at 5 muM strand concentration at room temperature. However, the immobile HJ constructed from four 18-mers was less stable, and four 12-mers did not form the HJ structure under the conditions used. A comparison of the melting profiles of the HJs with those of the duplexes corresponding to the arms of four-way junctions indicated that the thermal stability of the HJ was similar to that of the individual arm and the cooperativity of the melting behavior of the HJ was relatively higher than that of the individual arm duplex. The Tms of the mobile HJs containing 4, 6, 8, and 10 base-pair homologous cores at junctions were essentially identical with that of the immobile HJ of the same size. There is a tendency that the HJ containing a larger homologous core region becomes more resistant to thermal denaturation. The addition of divalent metal cations, Mg2+ and Ca2+, to the solutions of the HJs raised their melting temperatures. The difference found for the CD spectra of the HJs which differ only in the arrangement of the HJ depended primarily upon the DNA sequence flanking the junction. The RuvC protein binds to the immobile and mobile HJs, regardless of the presence and the size of the homologous core at the junction.