Two DNA ligase activities have been separated, purified, and characterized. The resolution of the two enzymes from crude extracts was initially achieved through Polymin P precipitation. The ligation activity precipitating with the nucleic acids on treatment with Polymin P is designated as DNA ligase I, and an activity remaining in the supernatant fraction, as DNA ligase II. DNA ligase I and II are ATP and Mg2+-dependent enzymes with pH optima of 7.8 and 8.0 and isoelectric points of 6.9 and 7.6, respectively. The purified I and II DNA ligase activities have molecular weights of 83,000 and 89,000, respectively. Both activities are inhibited by dATP and inorganic pyrophosphate. However, in the presence of optimum rATP levels, dATP stimulates DNA ligase II activity, whereas DNA ligase I is inhibited under the same conditions. Both activities are DNA specific and ligation follows reaction steps similar to those described for the Escherichia coli DNA ligase.