Human DHX9 helicase, also known as nuclear DNA helicase II (NDH II) and RNA helicase A (RHA), belongs to the SF2 superfamily of nucleic acid unwinding enzymes. DHX9 melts simple DNA-DNA, RNA-RNA, and DNA-RNA strands with a 3'-5' polarity; despite this little is known about its substrate specificity. Here, we used partial duplex DNA consisting of M13mp18 DNA and oligonucleotide-based replication and recombination intermediates. We show that DHX9 unwinds DNA- and RNA-containing forks, DNA- and RNA-containing displacement loops (D- and R-loops), and also G-quadruplexes. With these substrates, DHX9 behaved similarly as the RecQ helicase WRN. In contrast to WRN, DHX9 melted RNA-hybrids considerably faster than the corresponding DNA-DNA strands. DHX9 preferably unwound R-loops and DNA-based G-quadruplexes indicating that these structures may be biologically relevant. DHX9 also unwound RNA-based G-quadruplexes that have been reported to occur in human transcripts. It is believed that an improper dissolution of co-transcriptionally formed D-loops, R-loops, and DNA- or RNA-based G-quadruplexes represent potential roadblocks for transcription and thereby enhance transcription associated recombination events. By unwinding these structures, DHX9 may significantly contribute to transcriptional activation and also to the maintenance of genomic stability. Copyright © 2011 Elsevier B.V. All rights reserved.