DC-SIGN (dendritic cell-specific ICAM-3 grabbing nonintegrin), an external C-type lectin expressed on dendritic cells (DCs), has been proposed to play a pivotal role in trafficking HIV/SIV from mucosal surfaces to lymphoid tissues. Although the location of DC-SIGN expression has been established in a limited number of human tissues, its distribution in the rhesus macaque has not yet been determined. This study characterized the distribution and immunophenotype of DC-SIGN-expressing cells in SIV-infected and uninfected macaque tissues by immunohistochemistry (IHC) and confocal microscopy. IHC, using monoclonal and polyclonal antibodies against DC-SIGN, was performed on a variety of tissues. To further define the immunophenotype of DC-SIGN(+) cells, double-labeling with antibodies to CD68, fascin, and HLA-DR was done. In both infected and uninfected macaques, DC-SIGN(+) cells were located within the submucosa and lamina propria of tongue, vagina, rectum, and tonsil; however, no positive cells were present within the epithelium of any tissue. Antibodies to DC-SIGN also labeled Kupffer cells within the liver and scattered perivascular cells in the brain. Within lymph nodes, numerous positive cells were present within sinusoids in addition to cells consistent with interdigitating reticular cells in the paracortex and scattered follicular dendritic cells within germinal centers. In spleen of uninfected macaques, there was a similar distribution of DC-SIGN(+) cells with sinusoidal, marginal zone, and interdigitating dendritic cells staining; however, there was a marked paucity of staining in the spleens of SIV-infected macaques. DC-SIGN(+) cells were consistently CD68(+), but fascin(-) and HLA-DR(-). The absence of intraepithelial DC-SIGN-positive cells in mucosal tissues suggests that DC-SIGN does not play a significant role in transmucosal passage of HIV/SIV.