Neutralizing antibody assays are powerful tools for assessing humoral immunity in AIDS virus infection and vaccine development. Although many different assays have been described, all are based on the same principle, measuring reductions in virus infectivity. This unit describes two assays utilizing a genetically engineered cell lines that are susceptible to infection by most strains of HIV-1, SIV, and SHIV. One assay is designed for optimal performance with uncloned viruses produced in either PBMC or CD4+ T cell lines. A second assay is designed for single-cycle infection with molecularly cloned pseudoviruses produced by transfection in 293T cells. Both assays are performed in a 96-well format and use tat-responsive luciferase reporter gene expression as readout.