To gain further insights into the biological functions of Dna2, previously known as a cellular replicative helicase in Saccharomyces cerevisiae, we examined biochemical properties of the recombinant Dna2 protein purified to homogeneity. Besides the single-stranded (ss) DNA-dependent ATPase activity as reported previously, we were able to demonstrate that ssDNA-specific endonuclease activity is intrinsically associated with Dna2. Moreover, Dna2 was capable of degrading duplex DNA in an ATP-dependent fashion. ATP and dATP, the only nucleotides hydrolyzed by Dna2, served to stimulate Dna2 to utilize duplex DNA, indicating their hydrolysis is required. Dna2 was able to unwind short duplex only under the condition where the endonuclease activity was minimized. This finding implies that Dna2 unwinds only partially the 3'-end of duplex DNA and generates a stretch of ssDNA of limited length, which is subsequently cleaved by the ssDNA-specific endonuclease activity. A point mutation at the conserved ATP-binding site of Dna2 inactivated concurrently ssDNA-dependent ATPase, ATP-dependent nuclease, and helicase activities, indicating that they all reside in Dna2 itself. By virtue of its nucleolytic activities, the Dna2 protein may function in the maintenance of chromosomal integrity, such as repair or other related process, rather than in propagation of cellular replication forks.