From filtrates of an oxytetracycline-producing culture of Streptomyces rimosus a deoxyribonuclease was purified to homogeneity and determined to be a potent endo-DNase. It is a monomeric, basic protein (M(r) approximately 21,000; pI approximately 9.5) stable in a broad pH range but unstable to higher temperature. The enzyme has an absolute requirement for Mg2+ or Mn2+, and for its full activity requires free SH groups and a low-ionic-strength environment. Its N-terminal primary structure differs from that of other nucleases.