Microglial cells are central to brain immunity and intervene in many human neurological diseases. The aim of this study was to develop a convenient cellular model for human microglial cells, suitable for HIV studies. Microglia derive from the hematogenous myelomonocytic lineage, possibly as a distinct subpopulation but in any case able to invade the CNS, proliferate, and differentiate into ameboid and then ramified microglia in the adult life. We thus attempted to derive microglia-like cells from human monocytes. When cultured with astrocyte-conditioned medium (ACM), monocytes acquired a ramified morphology, typical of microglia. They overexpressed substance P and the calcium binding protein Iba-1 and dimly expressed class II MHC, three characteristics of microglial cells. Moreover, they also expressed a potassium inward rectifier current, another microglia-specific feature. These monocyte-derived microglia-like cells (MDMi) were CD4(+)/CD14(+), evocative of an activated microglia phenotype. When treated with lipopolysaccharide (LPS), MDMi lost their overexpression of substance P, which returned to untreated monocyte-derived macrophage (MDM) level. Compared with MDM, MDMi expressed higher CD4 but lower CCR5 levels; they could be infected by HIV-1(BaL), but produced less virus progeny than MDM did. This model of human microglia may be an interesting alternative to primary microglia for large scale in vitro HIV studies and may help to better understand HIV-associated microgliosis and chronic inflammation in the brain.