A 28-kDa ribonuclease, with an optimum pH of 4.0 and an optimum temperature at 58 degrees C, was isolated from fruiting bodies of the edible mushroom Hygrophorus russula. It was purified by ion exchange chromatography on carboxymethyl-cellulose, dithyaminoethyl-cellulose, quaternary amine-sepharose and sulphopropyl-sepharose, followed by fast protein liquid chromatography gel filtration on Superdex 75. The N-terminal amino acid sequence was ASAGG which showed homology to those of other fungal RNases to some degree. It exerted the highest RNase activity on poly C and poly U. The Michaelis constant (K-m) value of the RNase on yeast tRNA was 3.6 mu M, and the maximal velocity (V-max) was 0.6 mu M. The RNase activity was suppressed by some ions including Fe2+ and Zn2+ ions. The RNase inhibited the activity of HIV-1 reverse transcriptase with an IC50 of 4.64 mu M.