Eosinophils, when stimulated, release a variety of agents that can be toxic to ingested or extracellular targets. Among these systems is one that consists of eosinophil peroxidase (EPO), H2O2, and a halide. We report here that phorbol myristate acetate (PMA)-stimulated human eosinophils are virucidal to HIV-1 in a chloride-containing medium. When the eosinophil concentration is decreased to a level at which the virucidal effect is incomplete, the addition of bromide or iodide restored complete virucidal activity. The virucidal effect of eosinophils, PMA, and bromide under these conditions is inhibited by the peroxidase inhibitor azide and catalase, but not heated catalase or superoxide dismutase, implicating the EPO-H2O2-halide system. Purified EPO when combined with H2O2 in a chloride-containing medium is virucidal to HIV-1. When the EPO concentration is suboptimal, virucidal activity is increased by bromide, iodide, and, in this instance, thiocyanate and the virucidal activity of the bromide-supplemented system is inhibited by azide and catalase. Our findings, together with the demonstration that eosinophils express CD4 on their surface and, under some circumstances, can be productively infected with HIV-1, raise the possibility that biological oxidants formed by eosinophils can influence the pathogenesis of HIV-1 infection by their toxicity to eosinophil-associated or extracellular virus.