The rate of HIV-1 disease progression correlates strongly with plasma viral load and is likely to be influenced by both host and viral determinants. Though interest in the impact of viral replication capacity during HIV-1 infection has been increasing, especially with respect to drug resistance mutations, its influence on disease course remains poorly understood. This is due in part to significant drawbacks in conventional means of measuring HIV-1 growth in vitro (i.e. expense, inconvenience, and experimental variability). A FACS-based method is described here to measure HIV-1 replication sensitively and a modification of this method can be used to determine viral titer accurately. Importantly, the target cells used are permissive to CXCR4- and CCR5-tropic HIV-1 strains. In pilot experiments, the growth kinetics of laboratory-adapted strains NL4-3 and IIIB were examined carefully. Using this method, differences were observed in growth kinetics between three laboratory strains and seven primary isolates, indicating the potential for a broad range of in vitro replication capacities among individual isolates. In conclusion, this FACS-based method provides a sensitive approach to measure the replication capacity of HIV-1 and may prove useful in studies examining the impact of viral fitness on disease progression.