Ca/Mg-dependent nuclease is a possible key enzyme of apoptosis. We isolated and purified nucleases from the human and rat thymus to a homogeneous state and compared some properties of the obtained preparations with those of the earlier isolated Ca/Mg-dependent nuclease from the calf thymus. The activity of the nuclease from the human thymus in the presence of bivalent ions decreases in a sequence:(Ca + Mg) = (Ca + Mn) > Mn, that from the rat thymus: (Ca + Mn) > Mn > (Ca + Mg), and that from the calf thymus: (Ca + Mn) > (Ca + Mg) > Mn. Nuclease are not active in a medium containing only Mg, Ca, Co, and Zn ions. The preparations proved to be unstable during their isolation and storage. If the relative molecular mass of the purifies preparations was according to electrophoresis in 12% DS-Na-polyacrylamide gel 28, 29, and 18.4 and 21 kDa for the calf, human, and rat, respectively, after storage at -20 degrees C for two to six months, the molecular mass of native proteins decreases to 17-14 kDa. Some other properties of the enzymes have been described.