Human immunodeficiency virus type 1 (HIV-1) has evolved a number of strategies to resist current antiretroviral drugs and the selection pressures of humoral and cellular adaptive immunity. For example, R5 strains, which use the CCR5 coreceptor for entry and are the dominant viral phenotype for HIV-1 transmission and AIDS pathogenesis, are relatively resistant to neutralization by antibodies, as are other clinical isolates. In order to overcome these adaptations, we raised nucleic acid aptamers to the SU glycoprotein (gp120) of the R5 strain, HIV-1 Ba-L . These not only bound gp120 with high affinity but also neutralized HIV-1 infectivity in human peripheral blood mononuclear cells (PBMCs) by more than 1,000-fold. Furthermore, these aptamers were able to neutralize the infectivity of R5 clinical isolates of HIV-1 derived from group M (subtypes A, C, D, E, and F) and group O. One aptamer defined a site on gp120 that overlaps partially with the conserved, chemokine receptor-binding, CD4-induced epitope recognized by monoclonal antibody 17b. In contrast to the antibody, the site is accessible to aptamer in the absence of CD4 binding. Neutralizing aptamers such as this could be exploited to provide leads in developing alternative, efficacious anti-HIV-1 drugs and lead to a deeper understanding of the molecular interactions between the virus and its host cell.While current antiretroviral drugs have prolonged the quality of life for many human immunodeficiency virus type 1 (HIV-1)-positive individuals, they do not eliminate the virus (15, 37). The rapid emergence of drug-resistant HIV-1 strains has encouraged continued efforts to find novel antiretroviral agents with modalities different from those currently in use. One approach is to target the stage at which virus infects host cells. The entry of HIV-1 into target cells, its cellular tropism, and elements of the pathogenesis of AIDS are largely determined by the virion surface glycoprotein, gp120 (8,9,30). Variation in the hypervariable loops of gp120, particularly the V3 loop, determines the cellular tropism of the virus by governing the interaction with chemokine receptors such as CCR5 and CXCR4 (E. A. Berger, R. W. Doms et al., Letter, Nature 391:240, 1998). Strains that infect via CCR5, known as R5 strains, are preferentially transmitted from host to host (41), dominate the asymptomatic stage of infection (18,36), and are sufficient to cause AIDS (33).Elucidation of the three-dimensional crystal structure of gp120 (26, 27) coupled with site-directed mutagenesis (12, 24, 40) has revealed the remarkable way in which HIV-1 has evolved to protect its functionally conserved regions, thereby evading host antibody responses (26, 27). For example, regions of gp120 that interact with coreceptors are masked by extensive glycosylation and surface loops, limiting the ability of the immune system to mount a broad-spectrum neutralizing antibody response. These features partly explain the failure of candidate vaccine antigens based on recombinant HIV-1 surface envelope glycopro...