Quinoid precursors of melanin and/or reactive oxygen species (ROS) generated during melanogenesis have been implicated as cytotoxic molecules in the immune responses of insects against their internal metazoan parasites. No study has yet identified the killing components produced in conjunction with melanotic encapsulation responses, or explained how cytotoxic molecules generated in the open circulatory system of an insect can selectively destroy foreign tissues. Strains of Drosophila melanogaster with differing immune capabilities against the wasp parasitoid Leptopilina boulardi were examined for superoxide anion (O2-.) formation during parasitization. Elevated levels of O2-. were produced by immune reactive (R-strain) hosts during melanotic encapsulation of the parasitoid, but not by susceptible (S-strain) hosts in which the parasitoid developed unmolested. Both a superoxide dismutase (SOD)-deficient strain (cSODn108, red/TM3/Sb Ser) and a catalase (CAT)-deficient strain (Catn1) also produced melanotic capsules and elevated levels of O2-. when infected, but these reactions were unsuccessful and the parasitoids survived, indicating that neither the quinoid precursors of melanin nor O2-. per se were cytotoxic. Immune incompetence in SOD-deficient and CAT-deficient hosts is attributed in part to defects in hydrogen peroxide (H2O2) metabolism, and/or the inability of these metalloenzyme-deficient strains to initiate the metal-mediated reductive cleavage of H2O2 required for the production of the cytotoxic hydroxyl radical (.OH). The role proposed for O2-. in Drosophila cellular immunity is one of potentiating the formation of .OH. Melanin, which contains both oxidizing and reducing components, may serve a dual role in producing O2-. and sequestering redox-active metal ions, thereby confining the production of ROS. Host-parasite susceptibility in the Drosophila-Leptopilina system may be determined by the ability of the parasitoid to modulate hemocyte activity and prevent both effective melanotic encapsulation and the generation of cytotoxic levels of ROS.