Stonustoxin (SNTX) is a two-subunit protein purified from the venom of a stonefish, Synanceia horrida. It has potent lethal activity and is also a membrane pore-forming cytolysin. The role of thiol groups in the biological activities of SNTX was investigated. Both the hemolytic and lethal activities of SNTX were potentiated by the reducing agent, dithiothreitol (DTT). The hemolytic activity of SNTX was sensitive to the modification of thiol groups by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). The loss of haemolytic activity correlated with the number of thiol groups that were titrated with DTNB. Thiol modification of SNTX with DTNB also inhibited its lethality. These inhibitory effects of thiol modification could be reversed by reduction with DTT. It was also found that the haemolytic activity of SNTX could not be inhibited by cholesterol. These observations indicate that free thiol groups play an important role in the haemolytic activity and lethality of SNTX but unlike other thiol-activated cytolysins, SNTX was not inhibited by cholesterol. Thus, SNTX may represent a new class of cytolytic toxin.