Converging lines of research have linked human T-cell lymphotropic virus type III (HTLV-III) to the pathogenesis of the acquired immune deficiency syndrome. A characteristic feature of this virus is its genomic heterogeneity, which occurs to varying degrees in different viral isolates. To define further the nature and extent of these genomic changes, we compared the molecularly cloned genomes of two variant HTLV-III isolates by extensive restriction enzyme mapping and heteroduplex thermal melt analysis. Both viral isolates were found to be highly related to each other throughout their entire genomic complement, yet they differed markedly in their restriction enzyme maps. Electron microscopic heteroduplex analysis revealed several distinct regions of divergence located almost exclusively in the part of the genome that encodes the viral envelope gene. In vitro culture of one of these viruses over a period of 3 months did not result in any genomic changes as determined by restriction analysis of viral DNA. These results, as well as the recently published nucleotide sequences of other HTLV-III isolates, indicate that the most substantial variation among HTLV-III isolates is located in the envelope. These findings raise the possibility that viral isolates from different individuals could have important biological differences in their envelope antigens, a consideration relevant to ongoing attempts to develop a vaccine against HTLV-III.