RecA protein forms filaments on both single- and double-stranded DNA. Several studies confirm that filament extension occurs in the 5' to 3' direction on single-stranded DNA. These filaments also disassemble in an end-dependent fashion, and several indirect observations suggest that the disassembly occurs on the end opposite to that at which assembly occurs. By labeling the 5' end of single-stranded DNA with a segment of duplex DNA, we demonstrate unambiguously that RecA filaments disassemble uniquely in the 5' to 3' direction.