HIV-1 strains that use CCR5 predominate after transmission and during the asymptomatic period of disease. However, in up to half of infected people, variants that use CXCR4 emerge, coincident with accelerated disease progression. The earliest CXCR4 using strains to appear, called R5X4 viruses, usually retain CCR5 use. Prototype R5X4 HIV-1 isolates infect macrophages using CCR5 and CXCR4, but CD4+ lymphocyte infection by these viruses is mediated predominantly by CXCR4. Here, we sought to identify obstacles to CCR5 use on CD4+ lymphocytes by R5X4 HIV-1. Using a panel of R5X4 Envs we found that, although CXCR4 was the predominant coreceptor used to infect CD4+ lymphocytes, there was a spectrum of CCR5 use. Greater CCR5 use on lymphocytes correlated with relative resistance to inhibition by CCR5 mAbs and small molecule antagonists in CCR5+ indicator cells. Increasing CCR5 expression on primary lymphocytes through cytokine stimulation or lentiviral transduction increased the proportion of entry mediated by CCR5 for all R5X4 isolates except 89.6. Env dependence on CCR5 density was then evaluated using a cell line in which levels of CD4 and CCR5 could be independently regulated. At non-limiting CD4 levels, strains with greater lymphocyte CCR5 use were better able to exploit limiting levels of CCR5, whereas those that used lymphocyte CCR5 poorly were more sensitive to reductions in CCR5 levels. Evaluation of the V3 sequences of the R5X4 viruses using algorithms that ...