Opiate abuse has been postulated to be a cofactor in the immunopathogenesis of acquired immunodeficiency syndrome (AIDS). This study evaluated whether methadone, a drug widely prescribed for the treatment of drug abusers with opioid dependence, affects human immunodeficiency virus (HIV) infection of human immune cells. When added to human fetal microglia and blood monocyte-derived macrophage cultures, methadone significantly enhanced HIV infection of these cells. This enhancement was associated with the up-regulation of expression of CCR5, a primary coreceptor for macrophage-tropic HIV entry into macrophages. Most importantly, the addition of methadone to the cultures of latently infected peripheral blood mononuclear cells from HIV-infected patients enhanced viral activation and replication. Although the in vivo relevance of these findings remains to be determined, the data underscore the necessity of further studies to define the role of opioids, including methadone, in the immunopathogenesis of HIV infection and AIDS.