Sera from highly selected HIV-1-positive patients are known to have the ability to neutralize a diverse array of primary isolates of HIV-1. The human osteosarcoma cell line that expresses CD4 and chemokine receptors (GHOST cells) was adapted to study HIV-1 neutralization in 37 HIV-1-infected individuals who were selected because of slow disease progression or nonprogression. Many of these individuals were receiving combination drug therapy. Molecularly cloned HIV-1 JR-FL and NL4-3 viruses were used as prototypes to define assay conditions. Sera were then tested at a 1:40 dilution against six additional primary isolates, three of which utilized CCR5 and three of which used both CCR5 and CXCR4. The assay was highly reproducible and independent of viral input titer, with a readout at 48 hr equivalent to that at later time points. As previously reported, neutralization sensitivity was entirely independent of coreceptor usage. Only a few sera from slow progressors were able to neutralize a broad array of primary isolates at a 1:40 dilution, and the best clinical predictor of broadly neutralizing antibody for primary isolates was the present use of antiretroviral agents. In further studies it was found that purified antibody accounted for the majority of the measured neutralization. However, experiments with exogenous addition of antiviral agents showed that the use of nucleosides also greatly contributed to the measured neutralization in some patients. Measurement of neutralization of HIV-1 primary isolates by sera from patients receiving antiretroviral therapy must be carried out with some caution.