Primate lentiviruses such as human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) are phenotypically diverse, and virus isolates vary in cytopathicity, replication rate, and cell tropism. While all virus isolates infect primary peripheral blood lymphocytes, only a subset of strains infect established CD4-expressing T-cell lines. Here, we describe the development and characterization of a macaque cell line that can be infected by all of the strains of SIV that we have tested, including macrophage- and T-cell-tropic strains, primary and cell-line adapted strains, and SIVmac, SIVMne, and SIVsm isolates. The cells can be infected by strains of HIV type 2 (HIV-2) to varying degrees, but not by either cloned or primary isolates of HIV type 1 (HIV-1). This cell line is a derivative of a rhesus macaque mammary tumor cell line (CMMT) engineered to express human CD4. For these studies, a CMMT-CD4 clone expressing an integrated copy of a truncated HIV-1 long terminal repeat fused to the beta-galactosidase gene (LTR-beta-gal) was established to allow detection of infectious SIV after a single round of replication. Here, we demonstrate the ability of the CMMT-CD4-LTR-beta-gal cell line to rapidly and quantitatively detect infectious SIV. Using these cells to assay virus, we could readily measure neutralizing antibody activity in animals infected with different SIV isolates. Neutralizing activity was detected against the homologous virus and lower, but detectable, activity was measured against heterologous virus. Thus, this system, which is highly sensitive and can detect infection by all of the SIV isolates we tested, is a rapid method for detecting infectious virus and quantitating neutralizing antibody activity.