Strand exchange between two duplexes is usually initiated as a three-strand event that requires the presence of a single-stranded overhang or gap in one of the two molecules. Here we show that the RecA protein can catalyze a four-strand exchange. Specifically, it can recombine short hairpin substrates with homologous stems provided that one of the hairpins possesses a chimeric DNA/RNA backbone. This four-strand exchange reaction goes to completion in the presence of ATPgammaS and releases a stable heteroduplex upon removal of the RecA protein. Under identical conditions, strand exchange between two DNA hairpins is incomplete and generates a nascent heteroduplex that rapidly dissociates when the RecA protein is denatured. Since presynaptic filament formation does not appear to melt either type of hairpin, we propose that exchange occurs between homologously aligned duplexes that are extended and unwound within a RecA filament. The first reaction provides a mechanism for gene targeting by chimeric double-hairpin oligonucleotides while the second reaction explains the ability of the RecA protein to transiently align double-stranded DNA molecules.