DNA laddering is one of the biochemical processes characteristic of programmed cell death (PCD) both in animals and plants. However, the mechanism of DNA laddering varies in different species, even in different tissues of one organism. In the present study, we used root tip cells of rice, which have been induced by NaCl stress to undergo PCD, to analyze the endonuclease activities of cytoplasmic and nuclear extracts. Two endonucleases, a cytoplasmic of 20 kDa (OsCyt20) and a nuclear of 37 kDa (OsNuc37), were identified as PCD related. Our results indicated that OsCyt20 is a Ca2+/Mg2+-dependent nuclease, which is most active at neutral pH, and that OsNuc37 is Zn2+-dependent, with a pH optimum of 4.5-6. Both nucleases were induced at the early stage of PCD (2h salt treatment) and exhibited the highest activity approximately 4 h after exposure to NaCl, paralleling with the occurrence of DNA laddering. In vitro assays of endonuclease activities further revealed that OsNuc37, a glycoprotein localized in the nucleus, is the executor for DNA laddering. The different effects of both endonucleases on DNA degradation during salt-induced PCD are discussed.