A method for purification of beef spleen exonuclease is described, leading to electrophoretically homogeneous enzyme preparation. The method consists of three step fractionation of crude enzyme (after ammonium sulfate precipitation) as follows - ion exchange chromatography on ECTEOLA-cellulose, affinity chromatography on Concanavalin A-Sepharose and molecular sieving. The enzyme thus obtained is practically free of any contaminating activities - endonuclease or phosphomonoesterase. The molecular weight of the exonuclease was determined (98 000 +/- 3 000 daltons) and some other parameters of the enzyme were calculated. The investigation of the pH and thermo-stabilities showed significantly narrow limits of the exonuclease activity. The effect of the urea on the enzyme activity has also been evaluated.