Homing endonucleases are highly specific DNA-cleaving enzymes that recognize and cleave long stretches of DNA. The engineering of these enzymes provides instruments for genome modification in a wide range of fields, including gene targeting. The homing endonuclease I-SceI from the yeastSaccharomyces cerevisiaehas been purified after overexpression inEscherichia coliand its crystal structure has been determined in complex with its target DNA. In order to evaluate the number of ions that are involved in the cleavage process, thus determining the catalytic mechanism, crystallization experiments were performed in the presence of Mn2+, yielding crystals that were suitable for X-ray diffraction analysis. The crystals belonged to the orthorhombic space groupP212121, with unit-cell parametersa= 80.11,b= 80.57,c= 130.87 Å, α = β = γ = 90°. The self-rotation function and the Matthews coefficient suggested the presence of two protein–DNA complexes in the asymmetric unit. The crystals diffracted to a resolution limit of 2.9 Å using synchrotron radiation. From the anomalous data, it was determined that three cations are involved in catalysis and it was confirmed that I-SceI follows a two-metal-ion DNA-strand cleavage mechanism.