The Saccharomyces cerevisiae RAD3 gene, which is required for cell viability and excision repair of damaged DNA, encodes an 89-kDa protein that has a single-stranded DNA-dependent ATPase activity. We now show that the RAD3 protein also possesses a helicase activity that unwinds duplex regions in DNA substrates constructed by annealing DNA fragments of 71-851 nucleotides to circular, single-stranded M13 DNA. The DNA helicase activity is dependent on the hydrolysis of ATP, has a pH optimum of approximately 5.6, and is inhibited by antibodies raised against a truncated RAD3 protein produced in Escherichia coli. The RAD3 helicase translocates along single-stranded DNA in the 5'----3' direction. The direction of RAD3 helicase movement is consistent with the possibility that it unwinds DNA duplexes in advance of the replication fork during DNA replication.