A nucleic acid-dependent ATPase was purified from vaccinia virions and shown to have both DNA:DNA and RNA:RNA helicase activities. This is only the third helicase to be identified that can unwind both DNA and RNA duplexes. The DNA helicase activity copurified with nucleoside triphosphate phosphohydrolase II (NPHII), an RNA helicase encoded by gene I8R (S. Shuman, Proc. Natl. Acad. Sci. USA 89:10935-10939, 1992). Immunodepletion with two antisera to NPHII and analysis of recombinant NPHII protein (C. H. Gross and S. Shuman, J. Virol. 69:4727-4736, 1995) confirmed that the DNA helicase activity was encoded by the I8R gene. The I8R DNA helicase unwound DNA in a 3'-to-5' direction only, unwound duplexes of 35 bp but not 45 bp, and could be stimulated to unwind longer duplexes by the Escherichia coli single-stranded DNA-binding protein. DNA helicase activity was not stimulated by salt and was sensitive to 100 mM NaCl or KCl. The I8R protein has amino acid similarity to human RNA helicase A and to nuclear DNA helicase II, a bovine DNA and RNA helicase. On the basis of the phenotype of I8R temperature-sensitive mutants, it was suggested that the I8R protein is not required for DNA replication but might aid in the extrusion of early mRNA from the virus core. The DNA helicase activity of the I8R protein allows another interpretation of the mutant phenotype, namely, that the I8R DNA helicase activity is required for initiation of early transcription from within vaccinia virions.