A new exocytoplasmic, nutritionally controlled endodeoxyribonuclease (EC 3.1.21.-) was purified to homogeneity from Streptomyces antibioticus. The enzyme showed an apparent molecular mass of 29 kDa (being active in the monomeric form) and a pI of approximately 7.8. The nuclease hydrolysed endonucleolytically double-stranded circular and linear DNA. The enzyme makes nicks in one strand of the DNA in G-rich regions, leaving either 5' or 3' short, single-stranded overhangs with 3'-hydroxy and 5'-phosphate termini. Breaks in the DNA occur when two nicks in opposite strands are close together. The enzyme had an optimum pH of 7.5 and an absolute requirement for bivalent cations and > or = 100 mM NaCl in the reaction buffer. Activity was greatly diminished in the presence of phosphate, Hg2+ or iodoacetate and was stimulated by dimethyl sulphoxide. Single-stranded DNA was a much poorer substrate than double-stranded DNA. The nuclease hydrolyses sequences of three or preferably more (dG).(dC) tracts in the DNA. The initial specificity shifts to other sequences (including sequences shorter than those initially hydrolysed) during the course of the reaction, giving the changing pattern of bands observed in agarose gels. 5-Methylcytosine-hemimethylated DNA is not hydrolysed by the nuclease. The properties of this novel enzyme suggest a relationship with class II restriction endonucleases and also with some eukaryotic nucleases.