Two unique DNA fragments were recently identified in over 90% of acquired immunodeficiency syndrome (AIDS)-related Kaposi's sarcoma tissues. Sequence analysis suggests that these fragments belong to a previously unidentified human herpesvirus, Kaposi's sarcoma-associated herpesvirus (KSHV). These fragments have also been identified in a subset of non-Hodgkin's lymphomas in human immunodeficiency virus-positive patients; specifically, in body cavity-based lymphomas (AIDS-BCBLs). We have established two cell lines derived from AIDS-BCBLs, BC-1 and BC-2, which retain the KSHV sequences, and have used them to further characterize this putative viral genome. In this report, we demonstrate that the KSHV sequences represent a portion of a much larger DNA molecule that is located predominantly in the nucleus of the infected cells. In situ hybridization of metaphase spreads made from both of these cell lines show these sequences in episomal structures. Their presence in the cells as large nuclear episomes supports previous sequence homology data suggesting that KSHV belongs to the herpesvirus family. These cell lines provide a continuous source of KSHV sequences. Thus, they represent an important tool for future studies of this recently described human herpesvirus that may be involved in the pathogenesis of Kaposi's sarcoma and some AIDS-related non-Hodgkin's lymphomas.