The effects of (+)-beta-D-dioxolane-cytosine ((+)-D-beta-DOC), (-)-beta-L-dioxolane-cytosine ((-)-L-beta-DOC), (+)-beta-D-oxathiolane-cytosine ((+)-D-beta-OTC), (-)-beta-L-oxathiolane-cytosine ((-)-L-beta-OTC, or 3TC), 3'-azido-2',3'-dideoxy-5-methyl-cytidine (5-Me-AZDC), and 3'-azido-2',3'-dideoxyuridine (AZDU) on Epstein-Barr virus (EBV) DNA replication in vitro were tested in P3HR-1 cells. Two anti-EBV drugs, 3'-azido-3'-deoxythymidine (AZT) and 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG, or ganciclovir), were used as positive controls. The inhibitory effects on EBV DNA synthesis were quantified by membrane filter and Southern blot hybridizations with an EBV-specific probe BamHI-W fragment. The 50% effective doses (ED50) for EBV DNA replication were 0.15, 0.83, 1.5, 8.3, 14, and 7.7 microM for DHPG, (-)-L-beta-DOC, (+)-D-beta-DOC, (+)-D-beta-OTC, (-)-L-beta-OTC, and AZT, respectively. In contrast, 5-Me-AZDC and AZDU were not effective at concentrations as high as 30 microM. These results indicated that both (-)-L-beta-DOC and (+)-D-beta-DOC were more potent than AZT, which has previously been shown to have anti-EBV activity. (-)-L-beta-DOC and (+)-D-beta-DOC have also been previously demonstrated to suppress the infectivity of human immunodeficiency virus type 1 (HIV-1). Thus, (-)-L-beta-DOC represents the first nucleoside analog with L-configuration exhibiting significant antiviral activities against both EBV and HIV.