Cells of macrophage lineage represent a key target of human immunodeficiency virus (HIV) in addition to CD4-lymphocytes. The absolute number of infected macrophages in the body is relatively low compared to CD4-lymphocytes. Nevertheless, the peculiar dynamics of HIV replication in macrophages, their long-term survival after HIV infection, and their ability to spread virus particles to bystander CD4-lymphocytes, make evident their substantial contribution to the pathogenesis of HIV infection. In addition, infected macrophages are able to recruit and activate CD4-lymphocytes through the production of both chemokines and virus proteins (such as nef). In addition, the activation of the oxidative pathway in HIV-infected macrophages may lead to apoptotic death of bystander, not-infected cells. Finally, macrophages are the most important target of HIV in the central nervous system. The alteration of neuronal metabolism induced by infected macrophages plays a crucial role in the pathogenesis of HIV-related encephalopathy. Taken together, these results strongly support the clinical relevance of therapeutic strategies able to interfere with HIV replication in macrophages. In vitro data show the potent efficacy of all nucleoside analogues inhibitors of HIV-reverse transcriptase in macrophages. Nevertheless, the limited penetration of some of these compounds in sequestered districts, coupled with the scarce phosphorylation ability of macrophages, suggests that nucleoside analogues carrying preformed phosphate groups may have a potential role against HIV replication in macrophages. This hypothesis is supported by the great anti-HIV activity of tenofovir and other acyclic nucleoside phosphonates in macrophages that may provide a rationale for the remarkable efficacy of tenofovir in HIV-infected patients. Non-nucleoside reverse transcriptase inhibitors (NNRTI) do not affect HIV-DNA chain termination, and for this reason their antiviral activity in macrophages is similar to that found in CD4-lymphocytes. Interestingly, protease inhibitors (PIs), acting at post-integrational stages of virus replication, are the only drugs able to interfere with virus production and release from macrophages with established and persistent HIV infection (chronically-infected cells). Since this effect is achieved at concentrations and doses higher than those effective in de-novo infected CD4-lymphocytes, it is possible that lack of adherence to therapy, and/or suboptimal dosage leading to insufficient concentrations of PIs may cause a resumption of virus replication from chronically-infected macrophages, ultimately resulting in therapeutic failure. For all these reasons, therapeutic strategies aimed to achieve the greatest and longest control of HIV replication should inhibit HIV not only in CD4-lymphocytes, but also in macrophages. Testing new and promising antiviral compounds in such cells may provide crucial hints about their efficacy in patients infected by HIV.