Objective:To characterize HIV‐1 quasispecies at high resolution to determine thein vivosequence heterogeneity of virus infecting the brain.Methods:A 1 kilobase region of the envelope gene, which includes the five hypervariable regions, was amplified by polymerase chain reaction (PCR) using DNA obtained from brain tissue of an HIV‐1‐infected patient. PCR products were cloned and 50 clones sequenced.Results:Thirty‐nine unique nucleotide sequences producing 35 protein variants were found. A consensus sequence was identified along with three distinct subtypes, each present at a level of 12%. The sequence variation from the concensus was 0.1‐2.1% at the nucleotide level with hypermutation and recombination responsible for the highest diversity. Sequence heterogeneity resulted in both the creation and the elimination of N‐linked glycosylation sites. Only nine clones differed from the consensus sequence in the V3 loop. No inactivating mutations were found.Conclusions:HIV‐1 proviruses found in the brain generally demonstrate a low level of genetic variability inenv.However, genomes that vary considerably from the predominant species can be present at significant levels. This observation may be of importance for understanding viral pathogenesis in the central nervous system.AIDS 1995,9:345‐349