An endonuclease present in partially purified preparations of calf thymus DNA polymerase has been purified to homogeneity. It has a molecular weight of 53,000 +/- 2,500 as determined by sucrose gradient sedimentation. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicates the protein is composed of four subunits, each polypeptide possessing a molecular weight of 13,000. Its isoelectric point is 10.3 +/- 0.2. The endonuclease has a pH optimum at 6.6, requires Mg2+ or Mn2+ for activity, and does not attack RNA. The enzyme appears to be present in tissues other than calf thymus. The enzyme catalyzes the endonucleolytic cleavage of both denatured and native eukaryotic DNA. The enzyme introduces a limited number of single strand nicks into native DNA; hydrolysis of denatured DNA produces acid-soluble oligonucleotides. The average size of the limit product, sedimented in an alkaline sucrose gradient, is 1200 nucleotides for native DNA. The product contains 5'-phosphoryl and 3'-hydroxyl termini. While all four deoxynucleotides are found at the 5' termini, pyrimidine residues predominate. Calf thymus DNase V degrades closed circular duplex SV40 DNA and glucosylated T4DNA but not poly(dA-dT). The rate of hydrolysis of homopolymers is: poly(dT) greater than poly(dA) greater than poly(dC) greater than poly(dG) in the presence of Mg2+, and poly(dT) greater than poly(dC) greater than poly(dA) = poly(dG) in the presence of Mn2+.