A new acid deoxyribonuclease (DNase) was purified from the cultured mycelia of Cordyceps sinensis, and designated CSDNase. CSDNase was purified by (NH₄)₂SO₄ precipitation, Sephacryl S-100 HR gel filtration, weak anion-exchange HPLC, and gel filtration HPLC. The protein was single-chained, with an apparent molecular mass of ca. 34 kDa, as revealed by SDS-PAGE, and an isoelectric point of 7.05, as estimated by isoelectric focusing. CSDNase acted on both double-stranded (ds) and single- stranded (ss) DNA, but preferentially on dsDNA. The optimum pH of CSDNase was pH 5.5 and its optimum temperature 55. The activity of CSDNase was not dependent on divalent cations, but its enzymic activity was inhibited by high concentration of the cation: MgC1₂ above 150 mM, MnCl₂ above 200 mM, ZnCl₂ above 150 mM, CaCl₂ above 200 mM, NaCl above 300 mM, and KCI above 300 mM. CSDNase was found to hydrolyze DNA, and to generate 3-phosphate and 5-OH termini. These results indicate that the nucleolytic properties of CSDNase are essentially the same as those of other well-characterized acid DNases, and that CSDNase is a member of the acid DNase family. To our knowledge, this is the first report of an acid DNase in a fungus.