The crystal structure of Serratia endonuclease has been solved to 2.1 A by multiple isomorphous replacement. This magnesium-dependent enzyme is equally active against single- and double-stranded DNA, as well as RNA, without any apparent base preference. The Serratia endonuclease fold is distinct from that of other nucleases that have been solved by X-ray diffraction. The refined structure consists of a central layer containing six antiparallel beta-strands which is flanked on one side by a helical domain and on the opposite side by one dominant helix and a very long coiled loop. Electrostatic calculations reveal a strongly polarized molecular surface and suggest that a cleft between this long helix and loop, near His 89, may contain the active site of the enzyme.