A DNA glycosylase that releases free hypoxanthine by selective cleavage of dIMP residues in polydeoxynucleotides and DNA containing this nonconventional nucleotide is present in Escherichia coli cell extracts. The partly purified enzyme, termed hypoxanthine-DNA glycosylase, does not require divalent cations or phosphate for activity, and it acts more efficiently on double-stranded than on single-stranded substrates. The enzyme has properties different from either uracil-DNA glycosylase or 3-methyladenine-DNA glycosylase and is present at normal levels in E. coli mutants deficient in either of the latter two DNA glycosylases.