The envelope glycoprotein (Env) gp120/gp41 is required for HIV-1 infection of host cells. Although in general it has been perceived that more Env gives rise to higher infectivity, the precise quantitative dependence of HIV-1 virion infectivity on Env density has remained unknown. Here we have developed a method to examine this dependence. This method involves 1) production of a set of single-cycle HIV-1 virions with varied density of Env on their surface, 2) site-specific labeling of Envspecific antibody Fab with a fluorophore at high efficiency, and 3) optical trapping virometry to measure the number of gp120 molecules on individual HIV-1 virions. The resulting gp120 density per virion is then correlated with the infectivity of the virions measured in cell culture. In the presence of DEAE-dextran, the polycation known to enhance HIV-1 infectivity in cell culture, virion infectivity follows gp120 density as a sigmoidal dependence and reaches an apparent plateau. This quantitative dependence can be described by a Hill equation, with a Hill coefficient of 2.4 ؎ 0.6. In contrast, in the absence of DEAE-dextran, virion infectivity increases monotonically with gp120 density and no saturation is observed under the experimental conditions. These results provide the first quantitative evidence that Env trimers cooperate on the virion surface to mediate productive infection by HIV-1. Moreover, as a result of the low number of Env trimers on individual virions, the number of additional Env trimers per virion that is required for the optimal infectivity will depend on the inclusion of facilitating agents during infection.Like other enveloped viruses, HIV-1 uses the trimer-of-hairpins mechanism to catalyze the fusion between viral and cell membranes, an essential step during the life cycle of all envel- (7), suggesting that more Env may indeed confer selective advantages in HIV-1 transmission. At the mechanistic level, however, how the Env copy number may enhance virion transmission is not well understood. Although greater amounts of Env have been observed to increase infectivity (8), the dependence of HIV-1 virion infectivity on Env density has not been precisely described or explained.In this paper, we introduce a quantitative approach to this problem. In our approach, we use a provirus clone that is env Ϫ together with a separate wild-type env plasmid (pEnv) to generate HIV-1 virions (9). It is essential that the provirus is env Ϫ so that we can titrate pEnv from low to high quantities (8) to generate a set of virions that are expected to carry varied densities of Env on their surface, limited by the pEnv inputs (10). Meanwhile, the resulting virions are only infectious for a single cycle. The infectivity of the virions can thus be correlated with the Env content of these virions that can be directly measured by singlemolecule techniques without the complications from multiple rounds of infection.To determine Env density on individual virions, we have recently developed a technique named optical trapping virometry (OTV)...