Sexually transmitted HIV-1 strains utilize the chemokine receptor CCR5 for viral entry and inhibitors targeting this coreceptor offer great promise for antiretroviral therapy. They also raise the question, however, whether viral variants exhibiting altered coreceptor interactions and resistance against these antiviral agents might still be pathogenic. In the present study, we analyzed a SIVmac239 envelope (Env) mutant (239DL) containing two mutations in the V3 loop which reduced viral entry via CCR5 by 10- to 20-fold, disrupted utilization of common alternative SIV coreceptors and changed the way Env engaged CCR5. To evaluate its replicative capacity and pathogenic potential in vivo we infected six rhesus macaques with 239DL. We found that 239DL replication was only slightly attenuated early during infection. Thereafter, a D324V change, which restored efficient CCR5 usage and coincided with 239wt-like levels of viral replication, emerged in two animals. In contrast, the viral geno- and phenotype remained stable in the other four rhesus macaques. Although these animals had about 100-fold reduced viral RNA loads relative to 239wt-infected macaques, they showed pronounced CD4 T-cell depletion in the intestinal lamina propria, and one developed opportunistic infections and died with simian AIDS. Thus, changes in the V3 loop that diminished CCR5 usage and altered Env interactions with CCR5 reduced the pathogenic potential of SIVmac in rhesus macaques but did not abolish it entirely.