Cytotoxic activity in extracts of pupae and adults of various kinds of butterflies and moths was tested in vitro against the human gastric carcinoma cell line, TMK-1, which was chosen as an example of human carcinoma cells. Among the species examined, cytotoxicity was limited to Pieris rapae, Pieris napi and Pieris brassicae. Activity was found down to a dilution of 1/10(4), while with the other butterflies and moths no activity was observed, even at 1/10(2). When the cytotoxicity of the three developmental stages, larvae, pupae and adults, of Pieris rapae was compared, the pupae showed the strongest activity, the IC50 against TMK-1 cells being at the 1/10(6) dilution. For larvae and adults, the respective IC50 values were at the 1/10(5) and 5/10(5) dilutions. The active principle in the pupae of Pieris rapae was found to be heat-labile and not extractable with organic solvents, but precipitated with ammonium sulfate and digested by proteases, suggesting that it is a protein. This cytotoxic factor was named pierisin.