Some properties of phosphodiesterase I from microsomes of rat intestinal mucosa were studied using the p-nitrophenyl ester of thymidine 5′-phosphate as substrate. The optimum pH was found to be 9.5–9.6 in several buffer systems. The activity was destroyed at temperatures of 63–68°, depending on the pH of the medium. Mg2+, Ca2+, Sr2+, Ba2+, and Ni2+ had a variable activating effect while Al3+, Cu2+, and Be were inhibitory. The enzyme was inhibited completely by 10−5 M EDTA or EGTA in the assay system used. The evidence obtained suggested phosphodiesterase I is a metal-requiring enzyme. Glycine, bicine, and tricine inhibited the enzyme at higher concentrations. Cysteine, 2-mercaptoethanol, and dithiothreitol were strongly inhibitory at a concentration of 10−3 M. The Km of the enzyme was 8.5 × 10−4 M. In 6 M urea 40% of the activity of the enzyme was lost irreversibly after 24 h at 4°. In 3 M urea there was an immediate and reversible mixed competitive–noncompetitive inhibition and the Km was increased seven to eight times.