A deoxyribonuclease (DNase) of pancreatic origin has been purified from extracts of the pyloric caeca from Atlantic cod (Gadus morhua L.). The crude extract was prepared by mincing frozen caeca tissue in equal volumes of buffer. The enzyme was isolated from the supernatant after streptomycin sulfate precipitation and centrifugation. The purification scheme further included chromatography on Q-Sepharose Fast Flow and hydroxyapatite columns. Affinity adsorption chromatography of the hydroxyapatite fraction on 8-(6-aminohexyl)-amino-5'-AMP-Sepharose, revealed an apparently homogeneous protein with molecular weight of 35,000 Da as judged by NaDodSO4-PAGE. In sum a 644-fold enzymatic enrichment and 3.5% total enzyme recovery was achieved. The cod enzyme resembles DNase I-type enzymes with an alkaline pH activity optimum and shows dependency for Mg(2+). The pI of the enzyme is 6.5 as determined by isoelectric focusing and DNase-zymography. Our findings suggest that the nuclease is a member of the cod's digestive enzymes secreted from the connective tissue surrounding the caeca.