1. The purification of a nuclease from rat-liver mitochondria is described. The mitochondria are rendered soluble by treatment with Triton X-100 and, after fractionation with ammonium sulphate and acetone, the active fraction is further purified by chromatography on DEAE-cellulose and Sephadex G-75 to give a purification of over 700-fold. 2. The purified enzyme was only very slightly contaminated with deoxyribonuclease II, phosphodiesterase and phosphomonoesterase. The individual activities of these enzymes did not exceed 0·1% of the activity of the liver nuclease. 3. The purified enzyme attacked RNA more rapidly than denatured DNA and hydrolysed native DNA more slowly than denatured DNA. 4. There is some evidence to suggest that the nucleolytic activity of the purified preparation towards native DNA, denatured DNA and RNA is associated with a single protein. 5. The enzyme is relatively labile but is stabilized in the presence of 20% (w/v) glycerol or 10mm-2-mercaptoethanol.