W e demonstrated previously that CD45RA⁺ CD4⁺ T cells are infected primarily by syncytium-inducing (SI) HIV-1 variants, whereas CD45RO⁺ CD4⁺ T cells harbor both non-SI (NSI) and SI HIV-1 variants. Here, we studied evolution of tropism for CD45RA⁺ and CD45RO⁺ CD4⁺ cells, coreceptor usage, and molecular phylogeny of coexisting NSI and SI HIV-1 clones that were isolated from four patients in the period spanning SI conversion. NSI variants were CCR5-restricted and could be isolated throughout infection from CD45RO⁺ CD4⁺ cells. SI variants seemed to evolve in CD45RO⁺ CD4⁺ cells, but, in time, SI HIV-1 infection of CD45RA⁺ CD4⁺ cells equaled infection of CD45RO⁺ CD4⁺ cells. In parallel with this shift, SI HIV-1 variants first used both coreceptors CCR5 and CXCR4, but eventually lost the ability to use CCR5. Phylogenetically, NSI and SI HIV-1 populations diverged over time. We observed a differential expression of HIV-1 coreceptors within CD45RA⁺ and CD45RO⁺ cells, which allowed us to isolate virus from purified CCR5⁺ CXCR4^– and CCR5^– CXCR4⁺ CD4⁺ cells. The CCR5⁺ subset was exclusively infected by CCR5-dependent HIV-1 clones, whereas SI clones were preferentially isolated from the CXCR4⁺ subset. The differential expression of HIV-1 coreceptors provides distinct cellular niches for NSI and SI HIV-1, contributing to their coexistence and independent evolutionary pathways.