Nuclear DNA helicase II (NDH II) was first isolated from calf thymus using a DNA-unwinding assay. Subsequently it has been shown to be a homologue of human RNA helicase A (RHA) and the maleless protein (MLE) from Drosophila. Accordingly, the protein possesses both DNA and RNA unwinding activities. Also, it can use all four NTPs or dNTPs to fuel the reaction. At its N-terminus it possesses two double-strand RNA binding domains (dsRBD I and II), while the C-terminus comprises an imperfect glycine (G)- and arginine (R)-rich repeat, a so-called RGG-box that preferably binds to ssDNA or ssRNA. Many proteins interact with NDH II both at its N- and C-terminus and thereby mediate transcriptional regulation, RNA processing, and transport, the DNA damage response and genome surveillance. The latter includes the histone variant gamma-H2AX and the Werner syndrome helicase (WRN). Here we describe experimental approaches to obtain mechanistic information about this important nuclear helicase.