A novel DNA helicase has been isolated from Saccharomyces cerevisiae. This DNA helicase co-purified with replication factor C (RF-C) during chromatography on S-Sepharose, DEAE-silica gel high performance liquid chromatography (HPLC), Affi-Gel Blue-agarose, heparin-agarose, single-stranded DNA-cellulose, fast protein liquid chromatography MonoS, and hydroxyapatite HPLC. Surprisingly, the helicase could be separated from RF-C by sedimentation on a glycerol gradient in the presence of 200 mM NaCl. The helicase is probably a homodimer of a 60-kDa polypeptide, which by UV cross-linking has been shown to bind ATP. It has a single-stranded DNA-dependent ATPase activity, with a Km for ATP of 60 microM. The DNA helicase activity depends on the hydrolysis of NTP (dNTP), with ATP and dATP the most efficient cofactors, followed by CTP and dCTP. The DNA helicase has a 5' to 3' directionality and is only marginally stimulated by coating the single-stranded DNA with the yeast single-stranded DNA-binding protein RF-A.