Characterization of the neutralizing interaction between antibody and virus is hindered by the nonsynchronized progression of infection in cell cultures. Discrete steps of the viral entry sequence cannot be discerned, and thus, the mode of antibody-mediated interference with virus infectivity remains undefined. Here, we magnetically synchronize the motion and cell attachment of human immunodeficiency virus type 1 (HIV-1) to monitor the progression of neutralization, both in solution and following virus attachment to the cell. By simultaneous transfer of all viral particles from reaction solution with antibody to the cell-bound state, the precise rate of neutralization of cell-free virus could be determined for each antibody. HIV-1 neutralization by both monoclonal and polyclonal antibody preparations followed distinct pseudo-first-order kinetics. For all antibodies, cell types, and HIV-1 strains examined, postattachment interference served a major role in the neutralizing effect. To monitor the progression of postattachment interference, we synchronized the entry process at initiation and measured the escape of cell-bound virus from antibody. We found that different antibodies neutralized the virus over different time frames during the entry phase. Virus was observed to progress through a sequence of shifting sensitivities to different antibodies during entry, suggested here to correlate with the exposure time of the target epitope on receptor-activated viral envelope proteins. Thus, by monitoring the progression of HIV-1 entry under synchronized conditions, we identify a new and significant determinant of antibody neutralization capacity, namely, the time frames for neutralization during the course of the viral entry phase.It is commonly accepted that antibodies (Abs) neutralize viruses by binding to the virion surface (22, 34). Indeed, good correlation exists between neutralizing capacity and binding affinity of the Ab to the target epitope (35, 39). However, the mode of Ab-mediated interference with virus infectivity remains undefined, largely due to the limited ability to monitor the neutralizing interaction between virus and Ab, in solution or following virus attachment to the cell surface. The diffusionlimited nature of the virus-cell interaction is central to this shortcoming of current in vitro systems.Viruses in solution behave as charged colloidal masses. Their motion is controlled by diffusion (1, 32), and their attachment to cells is primarily determined by electrostatic interactions with the charged cell surface (12). It is these coupled stochastic processes of cell encounter and attachment that constitute the rate-limiting steps to infection of cells in culture (1,20). Cell attachment progresses continuously, and thus, infection is initiated asynchronously, precluding step-by-step monitoring of viral events that precede or follow the attachment step.The lack of synchrony has challenged attempts both to characterize the dynamics of the viral entry sequence and to determine the mechanism and precise ...