The MER3 protein of Saccharomyces cerevisiae is required for crossover in meiosis and has been suggested to act at the initiation of homologous pairing and the resolution of Holliday junctions. The purified MER3 protein is a DNA helicase that translocates along single-stranded DNA in the 3' to 5' direction displacing annealed DNA fragments. Here, MER3 was found to be able to unwind various double-stranded DNA (dsDNA) substrates, including a 30-bp dsDNA with a 20-nucleotide 3'-overhang, a 30-bp dsDNA with a 20-nucleotide 5'-overhang, a 50-bp dsDNA with blunt ends, and a Holliday junction with 25-bp arms, each of which had a blunt end. Efficient unwinding of the 3'-overhang substrate appeared to initiate by the binding of MER3 to the 3' single-stranded tail in a reaction that required six or more unpaired bases. Unwinding of the blunt end and 5'-overhang substrates appeared to initiate at the blunt ends of these substrates. Unwinding of the Holliday junction was more efficient than the unwinding of the blunt and 5'-overhang substrates and was influenced by Mg(2+) concentrations that cause changes in the structure of the junction. Possible roles for Holliday junction unwinding in meiotic crossover are discussed.