An enzymatic activity that inserts purines into depurinated DNA was found in a soluble enzyme extract of Escherichia coli. This activity brings about the insertion of adenine and guanine into the appropriate apurinic sites in double-stranded DNA by using the corresponding deoxyribonucleoside triphosphates as the purine donors. Magnesium ions are required for this activity, it is inhibited by caffeine, and it does not act on depurinated single-stranded DNA. The insertion activity described here may represent a step in a repair mechanism, "base-insertion repair," whereby apurinic sites (which may occur in double-stranded DNA either due to the removal of damaged purines with specific glycosylases or by spontaneous depurination) are directly filled with the correct missing purine base.