A protein of molecular weight approximately 120,000 was isolated from cultured human fibroblasts and HeLa cells on the basis of its ability to bind specifically to apurinic DNA. After separation from apurinic endonuclease activity, the protein was found to incorporate purine, but not pyrimidine, bases specifically into depurinated DNA so as to protect the apurinic sites from alkali. Purine base insertion activity was sensitive to heating and freezing as well as to caffeine and EDTA; it required K+ but not a divalent cation. Guanine, but not adenine, was incorporated into depurinated poly(dG-dC), whereas adenine, but not guanine, was incorporated into poly(dA-dT). After incorporation into depurinated DNA, guanine could be reisolated as dGMP. Although this activity suggests an alternative pathway for DNA repair that is independent of nucleotide excision, other functions for such an enzyme are possible.