Virion-free plasma from entomopoxvirus (MyseEPV)-infected larvae of the armyworm, Mythimna separata, contains a factor that adversely affects the survival of the gregarious braconid endoparasitoid, Cotesia kariyai. Heating or proteinase K treatment eliminates the toxic effect of virion-free plasma on the parasitoid, suggesting that the lethal factor is a protein. We purified the Protein Lethal to C. kariyai larvae (PLCK) from the virion-free plasma of MyseEPV-infected M. separata larvae by a three-step procedure using gel filtration and cation-exchange chromatography. Toxic activity was measured using an in vitro-cultured parasitoid bioassay. Parasitoid larvae cultured in IPL-41 medium (Weiss et al., In vitro 17 (1981), 495) containing 4.7 g/ml purified PLCK shrank and died within 3 days. The molecular weight of PLCK was estimated by sodium dodecyl sulfate?polyacrylamide gel electrophoresis to be about 28000, under both reducing and non-reducing conditions, indicating that in its native form the protein is a single 28-kDa polypeptide. Western blot analysis indicated that the lethal protein is not present in the hemolymph of uninfected host larvae, but is induced in the hemolymph by infection with MyseEPV. Western blot analysis also indicated that the proteins of virions and occlusion bodies of MyseEPV are not serologically related to PLCK. none