Abstract The activity of damage-dependent endonuclease in mouse plasmacytoma cells (line MPC-11) has been studied using damaged Φ × 174 RFI DNA as substrate. The DNA was treated with ultraviolet light, acid, or osmium tetroxide to introduce different types of lesions. Ultraviolet light-damaged DNA was cleaved at approx. 1.1 sites per 35 thymine-containing dimers by the extract, which indicates no specificity towards this type of lesion. The acid-treated DNA, which contains apurinic sites, was enzymatically broken in every alkalilabile site and this strongly suggests the presence of an apurinic-specific endonuclease activity in the nuclear extract. The activity which acts on ultraviolet-irradiated DNA and that which acts on acid-treated DNA have different specificities as shown by their salt requirements and the extent to which they are stimulated by magnesium. While the ultraviolet-endonuclease activity was very little affected by reducing the KCl concentration, the apurinic-specific activity was almost completely abolished. Osmium tetroxide renders the DNA an excellent substrate for endonucleolytic activity in the mouse cell extract. The response to KCl and MgCl 2 of the osmium tetroxide-specific endonuclease activity is qualitatively similar to that of the endonuclease activity, which acts on ultraviolet-irradiated DNA. Treatment of DNA with osmium tetroxide is known to produce 5,6-dihydroxy-dihydrothymine which is a minor photoproduct in DNA after irradiation, suggesting that the ultraviolet-specific endonuclease activity acts upon this lesion.