Immunodeficiency typically appears many years after initial HIV infection. This long, essentially asymptomatic period contributes to the transmission of HIV in human populations. In rare instances, clearance of HIV-1 infection has been observed, particularly in infants. There are also reports of individuals who have been frequently exposed to HIV-1 but remain seronegative for the virus, and it has been hypothesized that these individuals are resistant to infection by HIV-1. However, little is known about the mechanism of immune clearance or protection against HIV-1 in these high-risk individuals because it is difficult to directly demonstratein vivoprotective immunity. Although most of these high-risk individuals show an HIV-1-specific cell-mediated immune response usingin vitroassays, their peripheral blood lymphocytes (PBLs) are still susceptible to HIV infection in tissue culture. To study this furtherin vivo, we have established a humanized SCID mouse infection model whereby T-, B-, and natural killer-cell defective SCID/beige mice that have been reconstituted with normal human PBLs can be infected with HIV-1. When the SCID/beige mice were reconstituted with PBLs from two different multiply exposed HIV-1 seronegative individuals, the mice showed resistance to infection by two strains of HIV-1 (macrophage tropic and T cell tropic), although the same PBLs were easily infectedin vitro. Mice reconstituted with PBLs from non-HIV-exposed controls were readily infected. When the same reconstituted mice were depleted of human CD8 T cells, however, they became susceptible to HIV-1 infection, indicating that thein vivoprotection required CD8 T cells. This provides clear experimental evidence that some multiply exposed, HIV-1-negative individuals havein vivoprotective immunity that is CD8 T cell-dependent. Understanding the mechanism of such protective immunity is critical to the design and testing of effective prophylactic vaccines and immunotherapeutic regimens.