Human T-cell lymphotropic virus type III B (HTLV-III B) derived from H9/HTLV-III cells was cloned by a plaque-forming assay. Cloned viruses showed different restriction cleavage patterns, suggesting that HTLV-III B is a mixture of several viruses. Using two different neutralization assay systems, we compared the envelope antigenicity of uncloned HTLV-III B, cloned viruses, lymphadenopathy-associated virus (LAV), and acquired immune deficiency syndrome (AIDS)-associated retrovirus (ARV). A diversity of viral envelope antigens was consistently found among the strains of these AIDS-related retroviruses. These results concur with the previous finding that genomic divergence of AIDS-related viruses is located mainly in the region of the viral envelope gene. Thus, this plaque assay is useful for the clonal analysis of the virus, which is essential for comparing the envelope antigen(s) of the virus.