Abstract Three acid deoxyribonucleases designated O-I, O-II and O-III, were purified to varying degrees from the hepatopancreas of Octopus vulgaris by means of molecular sieve and ion-exchange chromatography. All three enzymes are endonucleases. Enzyme O-I, purified about 16-fold, has a pH optimum at 5.5, is accelerated by an addition of 0.025 m Mg +− and shows a slight preference for the d-Tp-Ap bond. Enzyme O-II, purified about 330-fold has a pH optimum at 6.0, has an absolute requirement for Mg +− (optimum at 0.02 m), shows a slight preference for the d-pT-pA bond and a significant preference for native DNA. Enzyme O-III, purified about 1400-fold is inhibited by the addition of Mg +−, has an optimum pH at 5.5 and manifests a slight preference for the d-Ap-Ap bond.