DC-SIGN, a type II membrane protein with a C-type lectin binding domain that is highly expressed on mucosal dendritic cells (DCs) and certain macrophages in vivo, binds to ICAM-3, ICAM-2, and human and simian immunodeficiency viruses (HIV and SIV). Virus captured by DC-SIGN can be presented to T cells, resulting in efficient virus infection, perhaps representing a mechanism by which virus can be ferried via normal DC trafficking from mucosal tissues to lymphoid organs in vivo. To develop reagents needed to characterize the expression and in vivo functions of DC-SIGN, we cloned, expressed, and analyzed rhesus macaque, pigtailed macaque, and murine DC-SIGN and made a panel of monoclonal antibodies (MAbs) to human DC-SIGN. Rhesus and pigtailed macaque DC-SIGN proteins were highly similar to human DC-SIGN and bound and transmitted HIV type 1 (HIV-1), HIV-2, and SIV to receptor-positive cells. In contrast, while competent to bind virus, murine DC-SIGN did not transmit virus to receptor-positive cells under the conditions tested. Thus, mere binding of virus to a C-type lectin does not necessarily mean that transmission will occur. The murine and macaque DC-SIGN molecules all bound ICAM-3. We mapped the determinants recognized by a panel of 16 MAbs to the repeat region, the lectin binding domain, and the extreme C terminus of DC-SIGN. One MAb was specific for DC-SIGN, failing to cross-react with DC-SIGNR. Most MAbs cross-reacted with rhesus and pigtailed macaque DC-SIGN, although none recognized murine DC-SIGN. Fifteen of the MAbs recognized DC-SIGN on DCs, with MAbs to the repeat region generally reacting most strongly. We conclude that rhesus and pigtailed macaque DC-SIGN proteins are structurally and functionally similar to human DC-SIGN and that the reagents that we have developed will make it possible to study the expression and function of this molecule in vivo.