The roles that human immunodeficiency virus (HIV) and Epstein-Barr virus (EBV) play in the genesis of acquired immune deficiency syndrome (AIDS)-related lymphomas are not understood. A human B cell line (B-HIV), developed to study AIDS-related lymphomagenesis, contains EBV and HIV genomes and is malignantly transformed. This line was produced by exposing B cells from an EBV-seropositive donor to HIV. To investigate the number of independent transformation events that took place at the time of HIV infection, we sought to determine how many transformed lineages are present in this cell line. B-HIV was found to have multiple different sites of HIV integration (> or = 25) as determined by fluorescence in situ hybridization. As a control, we analyzed a clonal cell line of HIV-infected human T cells, 8E5, and found HIV sequences located exclusively at band q22 in chromosome 13. We conclude that B-HIV is polyclonal, and viral sequences are located at multiple variable chromosomal sites in different B-HIV cells.