Sequence specific triple helix formation shows promise as a strategy for gene-specific inhibition of gene expression by blocking promoters or enhancers. Therefore, it is important to understand how this unusual structure affects DNA metabolic processes other than transcription. It has been shown that triplexes block in vitro DNA synthesis catalyzed by purified DNA polymerases. We report here that a purified DNA helicase unwinds a triple helical substrate with an efficiency similar to that observed with a comparable duplex species. These model studies suggest that triple helices will not seriously inhibit DNA replication or recombination in vivo, since DNA polymerases are preceded by helicases in the fully assembled replication holoenzyme.