The Okinawan sea anemone Actineria villosa causes severe cases of stinging. We isolated the 60 kDa A. villosa toxin (AvTX-60A) as the major toxin from the isolated nematocysts of this species. AvTX-60A showed fatal toxicity to mice with intraperitoneal injection at a minimum lethal dose of less than 250 microg/kg. The N-terminal amino acid sequence was determined and the corresponding cDNA encoding AvTX-60A was sequenced. The deduced amino acid sequence of AvTX-60A showed high similarity with PsTX-60A, which had been isolated as one of the major toxins from the venomous sea anemone Phyllodiscus semoni. These sea anemone toxins are new members of the family of proteins containing membrane-attack complex/perforin (MACPF) domains, best known in pore forming proteins such as perforin. These are the first examples of MACPF domain proteins as toxins for prey acquisition or repelling predators in nature.