A major endonuclease has been purified approximately 800-fold from rat liver nuclei using poly(A) as substrate. The enzyme had a molecular weight of about 50,000, and active fractions were obtained which contained no nucleic acid. Enzymatic activity was optimal between pH 6 and 7 and was totally dependent on the presence of a divalent cation. The reaction was inhibited by high ionic strength, polydextran sulfate, heparin, and sodium pyrophosphate. The purified enzyme readily hydrolyzed poly(A), poly(U), poly(C), and denatured DNA, whereas poly(G) was not degraded, and transfer RNA, ribosomal RNA, and native DNA were hydrolyzed only at relatively slow rates. These data suggest that the enzyme may be specific for single-stranded polynucleotides. The purified enzyme was essentially devoid of exonuclease activity, and the products of exhaustive endonuclease digestion of poly(A) were small oligonucleotides terminated with a 5'-phosphoryl group. Evidence was obtained that this endonuclease is localized in the nucleoplasm. Possible functions for this activity are discussed.