To date, attempts to eliminate HIV-1 infection from its latent reservoirs, a prerequisite for the development of a curative treatment strategy for HIV-1 infection, have been unsuccessful. We demonstrate that the FDA approved antifungal agent amphotericin B efficiently reactivates HIV-1 infection in THP89GFP cells, a model of HIV-1 latency in macrophages. Although amphotericin B does not directly reactivate latent HIV-1 infection in T cells (e.g., J89GFP), amphotericin-B-stimulated macrophages (THP89GFP cells or primary macrophages) when cocultured with J89GFP cells can induce HIV-1 reactivation in these cells in trans. Because of the close proximity of antigen presenting macrophages and T cells in the primary lymphoid organs, such interaction between antigen presenting macrophages and T cells are frequent, and it seems reasonable to assume that trans-reactivation strategies hold promise to also reactivate latent HIV-1 infection in vivo.