© 2015 Elsevier GmbH.Deoxyribonucleases (DNases) play a major role in apoptotic DNA fragmentation/degradation, and apoptotic-like DNA degradation is also observed during conjugation of the ciliate Tetrahymena thermophila; however, the characteristics of neutral and acidic DNases are still undefined in its life stages. Here, we report the biochemical characterization of DNase activities displayed in three different Tetrahymena life stages in a comparative manner. Maximum DNase activity of Tetrahymena was observed under acidic conditions, indicating that Tetrahymena has strong DNase II-like activities. Zymography revealed that Tetrahymena has at least five distinct DNase activity bands at 28, 32, 33.8, 35.5, and 69-kDa, and that the activities at 32 and 33.8-kDa were also secreted into starvation buffer. Cofactor analysis demonstrated that Mg2+ exerted inhibitory effects on neutral DNase activities. Unexpectedly, Mg2+ and Ca2+ had favorable effects on acidic DNase activities. The DNase activity profile of conjugating Tetrahymena cells revealed that the 32 and 33.8-kDa activities at pH 5.0 increased from 14 to 18h of conjugation, corresponding to the final resorption of the old macronucleus by lysosomal enzymes during programmed nuclear death (PND). Overall, we found that Tetrahymena DNases exhibit different biochemical properties and a possible involvement of DNase II-like activities in PND.