DNA replication of 2 human lymphoid cell lines (U296 and Raji), latently infected with the Epstein-Barr virus, has been compared using a density transfer approach. Typical of non-malignant lymphoblastoid cells, U296 cells divided once in bromodeoxyuridine-supplemented medium to form hybrid but not heavy-density host DNA. Replication of the intracellular Epstein-Barr virus DNA was selectively inhibited in these cells with only 15% of the viral genomes duplicating once to form hybrid-density viral DNA. However, some heavy-density viral DNA was formed in the U296 cells and DNA synthesis can thus initiate again on newly duplicated viral genomes in cells that have traversed only a single S phase. These results contrast strongly with observations concerning the Burkitt-lymphoma-derived cell line. Lymphoma cells are not growth-inhibited and most of the latent Epstein-Barr virus genomes of the Raji line replicated once, and only once, in successive S phases. While the majority of the 50 Epstein-Barr virus genomes of both the Raji and U296 cell lines are maintained as extra-chromosomal DNA plasmids, the control of their duplication is distinctly different in the respective malignant and non-malignant host cells.