This study examined the appearance of double-strand DNA breaks in rat brain after a focal cortical freeze injury in vivo. DNA fragments of oligonucleosome size appeared 3 h after the injury, and increased in a time-dependent manner. At 24 h, the amount of DNA fragmentation reached a maximum and then declined. When nuclei from freeze-injured brain tissue were incubated with Ca2+ in vitro, increased endonuclease activity, which can cause DNA fragmentation, was found. These findings indicate that the activation of a Ca(2+)-dependent endonuclease may be involved in the evolution of freeze-traumatized brain tissue.