An endo-exonuclease has been purified from cultured monkey (CV-1) cells. The enzyme which was purified to near homogeneity to be a 65 kDa monomeric protein. The single-strand DNase activity is endonudeolytic and nonprocessive, whereas the double-strand DNase activity is exonucleolytic and processive. The enzyme was also found to have RNase activity using poly-rA as substrate. The pH optimum for ss-DNase Is 8 and for ds-DNase it is 7.5. Both DNase activities require a divalent metal ion (Mg²⁺ , Mn²⁺ , Ca²⁺, Zn²⁺) for activity and exhibit the same kinetics of heat inactivation. The purified protein binds to and cleaves a synthetic Holliday junction substrate. The overall enzymatic characteristics of the mammalian protein are very similar to the putative recombination endoexonucleases purified from Neurospora crassa, Asperglllus nidulans and Saccharomyces cerevlslae.