To further document the role of toxic oxygen intermediates in mononuclear phagocyte antiprotozoal activity, microbicidal macrophages were depleted of the capacity to generate superoxide anion (O-2) and hydrogen peroxide (H2O2) by pretreatment with phorbol myristate acetate (PMA), a soluble agent which triggers the macrophage respiratory burst. Treating cells for 90 min with 200 ng/ml of PMA inhibited the extracellular release of both O-2 and H2O2 by 90% upon subsequent restimulation with either PMA or opsonized zymosan. This effect persisted for 48 h, and could not be reversed by the addition of lymphokine. Intracellular nitro-blue tetrazolium reduction by PMA-treated cells was also inhibited by 66-95% upon rechallenge with either PMA or inert or viable particulate agents. In parallel, PMA pretreatment abolished or markedly impaired the ability of normal, lymphokine-stimulated, and in vivo activated macrophages to kill three diverse protozoa--Toxoplasma gondii, Leishmania donovani, and Trypanosoma cruzi. These studies illustrate an additional technique for investigating the antiprotozoal effects of macrophage-derived O-2 and H2O2 and reemphasize the importance of an intact respiratory burst mechanism in killing of intracellular parasites.