The crystal structure of the periplasmic/extracellular endonuclease I from Vibrio salmonicida has been solved to 1.5 A resolution and, in comparison to the corresponding endonucleases from V. cholerae and V. vulnificus, serves as a model system for the investigation of the structural determinants involved in the temperature and NaCl adaptation of this enzyme class. The overall fold of the three enzymes is essentially similar, but the V. salmonicida endonuclease displays a significantly more positive surface potential than the other two enzymes owing to the presence of ten more Lys residues. However, if the optimum salt concentrations for the V. salmonicida and V. cholerae enzymes are taken into consideration in the electrostatic surface-potential calculation, the potentials of the two enzymes become surprisingly similar. The higher number of basic residues in the V. salmonicida protein is therefore likely to be a result, at least in part, of adaptation to the more saline habitat of V. salmonicida (seawater) than V. cholerae (brackish water). The hydrophobic core of all three enzymes is almost identical, but the V. salmonicida endonuclease has a slightly lower number of internal hydrogen bonds. This, together with repulsive forces between the basic residues on the protein surface of V. salmonicida endonuclease I and differences in the distribution of salt bridges, probably results in higher flexibility of regions of the V. salmonicida protein. This is likely to influence both the catalytic activity and the stability of the protein.