FEN-1 proteins are a family of nucleases essential for lagging strand DNA synthesis. A gene with sequence similarity to FEN-1 protein-encoding genes, rad2 +, has been identified in Schizosaccharomyces pombe . We report the overexpression, purification, and character-ization of the putative S.pombe FEN-1 homolog, Rad2p. A GST-Rad2p fusion protein was over-expressed in Saccharomyces cerevisiae and purified to near homogeneity by GST affinity chromatography. Although Rad2p had been previously classified as a putative FEN-1 protein based on amino acid homology, there has been no biochemical evidence demonstrating flap endonuclease activity. DNA cleavage analysis of several different oligodeoxynucleotide structuresindicates that GST-Rad2p possesses both 5'-flap endonuclease and 5'-->3' double-stranded DNA exo-nuclease activities. GST-Rad2p incises a 5'-flap and a 5'-pseudo-Y structure one base 3' of the branch point in the duplex region and also degrades double-stranded DNA. This is the first report on the biochemical characterization of S.pombe Rad2p. The potential roles of Rad2p in DNA excision repair and other nucleic acid reactions are discussed.