HIV infection results in a lifelong condition that when treated resembles a chronic disease. However, despite of years of fully suppressive therapy HIV persists in the host and is never eradicated. Key to HIV persistence is the infection of several different types of cells each of which has been postulated to be an important determinant of pathogenesis and persistence. Macrophages have been long considered to be key contributors to HIV infection and its dissemination into the CNS, a sanctuary somewhat independent of the periphery. However, recent studies have contradicted these early work and suggest that macrophages are not in vivo targets of HIV infection. To address this question we first determined the presence of HIV in monocytes isolated from viremic patients and from patients undergoing antiretroviral treatment. Consistent with these recent reports we failed to find viral DNA in blood monocytes from infected patients. Since the analysis of tissue macrophages is limited by the difficulties associated with invasive procedures we developed and implemented a novel humanized mouse model in which only myeloid cells are susceptible to HIV-1 infection. Using these myeloid only mice we demonstrate (1) that macrophages can sustain HIV replication in vivo in the absence of T cells, (2) that HIV infected macrophages are systemically distributed in all tissues analyzed including the brain, (3) that replication competent virus can be rescued from infected macrophages ex-vivo and (4) that macrophages can establish de novo infection in uninfected animals. These results demonstrate that macrophages represent a bone fide target for HIV infection in vivo that can sustain and transmit infection.