HepG2-derived hepatoblastoma cells (2.215), which actively produce hepatitis B virus (HBV), were cultured in the presence of 2',3'-dideoxyguanosine (ddG), 2',3'-dideoxyinosine (ddI), or 3'-azido 2',3'-dideoxythymidine (AZT). ddG was the most potent agent diminishing viral replication by as much as 95% as assessed by the amount of episomal HBV DNA without impairing cellular growth. AZT was least effective against HBV. Northern blot analysis revealed no apparent difference in the pregenomic viral RNA profile, suggesting that these dideoxynucleosides suppress reverse transcription in the replicative cycle of HBV. The effect of varying the time of drug exposure showed that these agents can suppress HBV replication even when added late in culture. HBV replication in another 2.2.15 cell population of the same lineage was affected by ddG differently, which may present an opportunity to investigate phenotypic and/or genetic alterations during culture. The present data suggest that some 2',3'-dideoxynucleosides can exert a potent antiviral activity against HBV in vitro at least under certain circumstances.