When HIV-infected leukocytes are activated by the CD28 costimulatory receptor, HIV-1 is rapidly cleared from cultures, suggesting that costimulation can render T cells resistant to HIV-1 infection. In this study we tested the hypothesis that enhanced secretion of cytokines or chemokines could account for CD28-induced antiviral effects. In an acute infection system, resistance to infection with macrophage-tropic strains of HIV-1 was shown to be comprised of both soluble and cell-associated components. Induction of HIV-1 resistance was specific for CD28 costimulation, in that a variety of other accessory receptors, such as CD2, CD4, CD5, and MHC class I, failed to confer the antiviral resistance. The soluble component was secreted by both CD4 and CD8 T cells, was not unique to CD28 costimulation, and could be neutralized by removal of C-C chemokines (RANTES (regulated upon activation, normal T cell expressed and secreted) and macrophage inflammatory protein-1alpha and -1beta) from the culture supernatants of costimulated CD4 T cells. In contrast, CD28 stimulation of CD4 cells resulted in the specific induction of a pronounced intrinsic resistance to HIV-1 infection by macrophage tropic isolates of HIV-1.