We used experimental infection of rhesus macaques with envelope gp120 V3 loop isogenic simian-human immunodeficiency virus (SHIV) molecular clones to more clearly define the impact of human immunodeficiency virus type 1 coreceptor usage in target cell selectivity and the rates of CD4⁺-T-cell depletion. Functional assays demonstrate that substitution of the V3 loop of the pathogenic CXCR4-tropic (X4) SHIV_SF33A2 molecular clone with the corresponding sequences from the CCR5-tropic (R5) SHIV_SF162P3 isolate resulted in a switch of coreceptor usage from CXCR4 to CCR5. The resultant R5 clone, designated SHIV_SF33A2(V3), is replication competent in vivo, infecting two of two macaques by intravenous inoculation with peak viremia that is comparable to that seen in monkeys infected with X4-SHIV_SF33A2. But while primary infection with the X4 clone was accompanied by rapid and significant loss of peripheral and secondary lymphoid CD4⁺ T lymphocytes, infection with R5-SHIV_SF33A2(V3) led to only a modest and transient loss. However, substantial depletion of intestinal CD4⁺ T cells was observed in R5-SHIV_SF33A2(V3)-infected macaques. Moreover, naïve T cells that expressed high levels of CXCR4 were rapidly depleted in X4-SHIV_SF33A2-infected macaques, whereas R5-SHIV_SF33A2(V3) infection mainly affected memory T cells that expressed CCR5. These findings in a unique isogenic system illustrate that coreceptor usage is the principal determinant of tissue and target cell specificity of the virus in vivo and dictates the dynamics of CD4⁺-T-cell depletion during SHIV infection.