Patricia E. Gallagher and Thomas P. Brent** abstract: A DNA glycosylase was isolated and purified over 1000-fold from human placentas by means of diethylaminoethylcellulose and double-and single-stranded DNA-Sepha-rose affinity chromatography. The procedure was rapid and yielded> 15% of the initial enzyme activity. High-pressure liquid chromatographs of reactionproducts showed that 3-methyladenine was the predominant substrate in methylated native DNA. 7-Methylguanine and 3-methylguanine were also released by the partially purified enzyme, albeit at low rates; release was more evident when the substrate was methylated double-stranded poly (dG-dC). The enzyme preparation was essentially free of nuclease activity, retaining< 0.001% of the initial cellularconcentration of Mg2+-requiring apurinic en-donuclease activity. Theglycosylase had a broad pH optimum between 7.2 and 7.7; it did not require metal ions but was