An endonuclease specific for apurinic sites in double-stranded DNA was partially purified from calf liver extracts. The enzyme has a pH optimum of 9.5, is only slightly stimulated by low concentrations of Mg2+, and has a MW of 28,000. Inhibitors of the endonuclease include Ca2+, EDTA, p-HOHgBzO [p-hydroxymercuribenzoate], NaCl and tRNA. The enzyme introduces single- and double-stranded breaks in depurinated DNA. High concentrations of the enzyme preparation degrade untreated single-stranded DNA, but not UV irradiated DNA or DNA treated with methylmethanesulfonate or 7-bromomethyl-12-methylbenz[a]-anthracene. Enzymatic incisions produce 3''-hydroxyl and 5''-phosphate end groups. Some of the properties of the calf liver apurinic endonuclease differ from those of a similar endonuclease obtained from calf thymus by S. Ljungquist and T. Lindahl and in this laboratory. These may be isozymes.