Intestinal macrophages, unlike macrophages from other tissues, do not support HIV-1 infection or produce proinflammatory cytokines. In vitro studies suggest this unique, functional phenotype is a result of the exposure of newly recruited blood monocytes to intestinal stromal products. However, in AIDS-related CMV colitis, mucosal macrophages express HIV-1 and proinflammatory cytokines. Therefore, we investigated the mechanism by which CMV confers permissiveness to HIV-1 and cytokine production on intestinal macrophages. We show that intestinal stroma-conditioned media (S-CM) down-regulated monocyte-derived macrophage infection by HIV-1 (pseudotyped with YU2 envelope or vesicular stomatitis virus glycoprotein) and production of TNF-α, but preinfection of the cells with CMV reversed this down-regulation, enhancing HIV-1 infection, p24 production, and TNF-α release. The ability of CMV to reverse S-CM down-regulation of macrophage HIV-1 infection was blocked by anti-TNF-α antibodies and over-ridden by exogenous TNF-α. Immunohistochemical analysis of monocyte-derived macrophages exposed to CMV and HIV-1 (YU2 pseudotype) revealed that the cells infrequently contained CMV and HIV-1 viral proteins. In addition, analysis of colon tissue sections from HIV-1-infected patients with CMV colitis showed that some macrophage-like cells contained CMV and TNF-α proteins, others contained HIV-1 and TNF-α proteins, but cells infrequently contained CMV and HIV-1 proteins. These results indicate that CMV blocks stromal product inhibition of HIV-1 infection in macrophages, and this inhibition is mediated, at least in part, by CMV-induced TNF-α acting in trans to enhance HIV-1 infection. © Society for Leukocyte Biology.