Immune hemolymph was collected from fifth instar larvae and 1-day-old pupae of Heliothis virescens after injection of prepupae with live Enterobacter cloacae. Induction of antibacterial activity against Escherichia coli K12 D31 was 7.5 times greater in pupal than in larval immune hemolymph. Lysozyme activity of immune pupal hemolymph against Micrococcus lysodeikticus was 11 times greater when compared with lysozyme activity of immune larval hemolymph. Early pupal immune response with regard to antibacterial activity was much greater than larval immune response in H. virescens. Normal pupal hemolymph showed an increase in antibacterial activity and lysozyme that was induced during metamorphosis. Antibacterial protein was isolated together with lysozyme by gel filtration chromatography and then separated from lysozyme by sequential electrophoresis with a native acid gel and SDS gel. Molecular mass of antibacterial protein was estimated to be 12 kDa. The N-terminal amino acid sequence of 12-kDa protein was different from those of antibacterial molecules found in other insects and has not been identified before. A sample containing 12-kDa protein was negative for immunoblotting with anti-synthetic cecropin B antibody. We have named the novel 12-kDa antibacterial protein viresin. Viresin showed antibacterial activity against several Gram-negative bacteria including E. cloacae but not against Gram-positive bacteria.