A deoxyribonuclease distinct from the previously isolated asparagus ribosome-inactivating proteins, possessing a molecular weight of 30 kDa and requiring a pH of 7.5 for optimum hydrolytic activity toward herring sperm DNA, was isolated from Asparagus officinalis seeds. The isolation procedure involved extraction with saline, (NH,,),SO, precipitation, ion-exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion-exchange chromatography on CM-Sepharose, and FPLC gel filtration on Superdex 75, The doxyribonuclease was unadsorbed onto DEAE- cellulose and Affi-gel blue gel and adsorbed onto CM-Sepharose. It exhibited the novel N-terminal sequence, GIEVIKIREL. The deoxyribonuclease was purified to a specific activity of 1584 units/mg. It was devoid of ribonuclease, protease, and HIV-1 reverse transcriptase-inhibitory activities. However, it inhibited cell-free translation in a rabbit reticulocyte lysate system with an IC50 of 20 muM. It exhibited antifungal activity toward Botrytis cinerea but not toward Fusarium oxysporum and Mycosphaerella arachidicola.