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Four hitherto undescribed endodeoxyribonucleases, temporarily designated A(1), A(2), A(3), and B, have been isolated from E. coli K-12. Each requires Mg(++) and is not stimulated by ATP or S-adenosylmethionine. A(3) is strongly inhibited by Fe(+++) and weakly inhibited by ATP, S-adenosylmethionine, and DPN, whereas B is inhibited by caffeine. Each can be purified free of exonuclease or DNA-3'-phosphatase. A(1) (molecular weight approximately 72,000) cleaves single-stranded, circular fd DNA to form 3'-hydroxyl termini and introduces nicks and breaks in the closed, double-stranded replicative form DNA of fd (fd RFI). A(2) (molecular weight approximately 46,000) cleaves fd DNA and introduces nicks and breaks in RFI, forming 3'-hydroxyl- and 5'-phosphoryl termini. A(3) (molecular weight approximately 38,000) cleaves fd DNA to form 3'-hydroxyl termini and introduces only nicks in fd RFI. Irradiation of the RFI with ultraviolet light markedly increases the rate of hydrolysis by A(3). B appears to form 3'-phosphoryl termini with fd DNA, but its characterization is highly preliminary due to its instability.