An endodeoxyribonuclease has been purified to near homogeneity from rat small intestinal mucosa by a procedure involving Con A-Sepharose affinity chromatography. During the initial steps of purification, the presence of 5 mM CaCl2 was essential for stability of the enzyme activity. The enzyme has a molecular weight of 32 000 and an isoelectric point of 4.7. NaCl, sulfhydryl reagents, and iodoacetate strongly inhibited the reaction, but tRNA did not. The enzyme required divalent cations for activity and had a pH optimum of pH 6.2 with Co2+ and pH 7.7 with Mn2+. In both optimum conditions, the enzyme hydrolyzed native DNA more rapidly than denatured DNA, and the average chain lengths of limit digestion products of native and denatured DNA were 8 and 10, respectively, at pH 6.2 and 9 and 11, respectively, at pH 7.7. The enzyme activity to produce acid-soluble fractions from linear DNA substrate was similar in the two optimum conditions, but the activity to nick double-stranded, superhelical circular DNA substrate was significantly higher at pH 6.2 than at pH 7.7. The endonuclease formed single-strand breaks making 5'-phosphoryl and 3'-hydroxyl termini, and deoxythymidine was present at the 5' termini with a frequency of about 50% in both optimum conditions. Bovine pancreatic DNase I antibody and G-action inhibited the enzyme activity. Thus this endonuclease is classified as a DNase I.