The properties of the deoxyribonuclease activity in the intestinal mucosa of rat have been studied. It was found that the activity was extractable from a whole cell preparation of the tissue with either Krebs Ringer phosphate buffer (pH 7.8) or physiological saline. The former was a better extracting medium, the buffered extract being more stable to storage at -20°C. Two active proteins were precipitated on fractionation of the Krebs Ringer phosphate buffer extract with ammonium sulfate. One fraction precipitated at 20% saturation of (NH₄)₂SO₄ was identified as DNase I by its optimum pH, ionic requirements and by its reaction to known DNase I inhibitors such as EDTA, citrate and arsenate. Ion-exchange chromatography on DEAE-cellulose (chloride form) established that the products of reaction ranged from mononucleotides through to oligonucleotides with a degree of polymerization larger than 7. These products were shown to carry a phosphomonoester linkage on the 5’-carbon. This fraction was designated as the 20%P enzyme and represented a 20 fold purification of the crude deoxyribonuclease extract. The supernatant fraction obtained after the 20%P enzyme was removed by centrifugatlon was found to still contain - i i - considerable DNase activity. This residual activity disappeared from the solution when the ammonium sulfate concentration was increased to 30% saturation, but all attempts to recover the DNase activity from the protein precipitated at this salt concentration were unsuccessful. Consequently, studies of this residual DNase activity were carried out using the crude extract. This second deoxyrlbonuclease was shown to be qualitatively different from the 20% enzyme. It did not require activation by Mg⁺⁺, and was not inhibited by EDTA, citrate or arsenate. Some of the products formed by the crude enzyme extract were shown to terminate in 3’-phosphoryl groups. These products were not formed by the 20%P enzyme and therefore must be due to the residual deoxyrlbonuclease. In all likelihood then, this second deoxyribonuclease activity was of the DNase II type. The intracellular distribution of the intestinal mucosal deoxyribonuclease was studied by differential centrifugation of the tissue homogenate. DNase I activity was found to occur in the mitochondria whereas DNase II was associated with some lighter subcellular particles. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate