Simian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV) gp160s obtained from the brain are often genetically distinct from those isolated from other organs, suggesting the presence of brain-specific selective pressures or founder effects that result in the compartmentalization of viral quasi-species. Whereas HIV has also been found to compartmentalize within different regions of the brain, the extent of brain-regional compartmentalization of SIV in rhesus macaques has not been characterized. Furthermore, much is still unknown about whether phenotypic differences exist in envelopes from different brain regions. To address these questions, env DNA sequences were amplified from four SIVmac239-infected macaques and subjected to phylogenetic and phenetic analysis. The authors demonstrated that sequences from different areas of the brain form distinct clades, and that the long-term progressing macaques demonstrated a greater degree of regional compartmentalization compared to the rapidly progressing macaques. In addition, regional compartmentalization occurred regardless of the presence of giant-cell encephalitis. Nucleotide substitution rates at synonymous and nonsynonymous sites (ds:dn rates) indicated that positive selection varied among envelopes from different brain regions. In one macaque, envelopes from some but not all brain regions acquired changes in a conserved CD4-binding motif GGGDPE at amino acids 382 to 387. Furthermore, gp160s with the mutation G383E were able to mediate cell-to-cell fusion in a CD4-independent manner and were more susceptible to fusion inhibition by pooled plasma from infected macaques. Reversion of this mutation by site-directed mutagenesis resulted in reduction of CD4-independence and resistance to fusion inhibition in cell fusion assays. These studies demonstrate that SIV evolution within the brain results in a heterogeneous viral population with different phenotypes among different regions.