Using a panel of amphotropic murine leukemia virus packaging cell lines that differed only in their levels of envelope protein (gp70) expression, we examined the relationship between transduction and the number of envelope proteins per virus. We generated virus stocks that contained different levels of virus-associated envelope proteins, purified them from gp70 that was not associated with the viruses, quantified their titers, and measured the efficiency with which they transduced NIH 3T3, TE671, and HeLa cells. As expected, titers increased monotonically with viral envelope protein number. Titers are measured using highly dilute virus, however, and are often not predictive of gene transfer when high doses of virus are used, as is done in gene therapy protocols. Interestingly, when we used high doses of virus, we observed significantly different trends: gene transfer increased, reached a maximum, and then declined sharply as the number of envelope proteins per virus increased. The highest levels of gene transfer occurred when cells were transduced with a moderate dose of virus that contained low levels of envelope protein. Our results indicate that transduction is inhibited when viruses that contain large numbers of envelope proteins are used. This is most likely because each virus, when it binds to a cell, delivers a large payload of envelope proteins that occupy or inactivate multiple virus receptors, reducing or eliminating the susceptibility of the cell to being transduced by additional viruses. The implications of our findings for the design of improved retroviral vectors for human gene therapy are discussed.