In this work, we investigated the anti-hepatitis B virus (HBV) activity of lamivudine, adefovir, tenofovir, penciclovir and lobucavir after short-term (i.e. 24 or 48 h) or continuous (9 days) exposure of the HBV-containing cell line, HepG2 2.2.15, to these drugs. Lamivudine maintained significant anti-HBV activity when added for only 24 or 48 h to the cell cultures compared to when the drug was present for the whole period (9 days) on the cells, i.e. 50% effective concentration (EC50) values for the inhibition of HBV DNA synthesis were 0.07 +/- 0.02 microgram ml-1 after 24 h of incubation, 0.02 +/- 0.01 microgram ml(-1) after 48 h of incubation and 0.0016 +/- 0.001 microgram ml(-1) after 9 days of incubation. Similarly, the nucleoside phosphonate analogues, adefovir and tenofovir, retained significant anti-HBV activity when added for only a short period of time to the cells. The EC50 values were 12 +/- 1 microgram ml(-1) (24 h) and 1.0 +/- 0.2 microgram ml(-1) (48 h) vs 0.003 +/- 0.001 microgram ml(-1) (9 days) for adefovir, and 6.5 +/- 1.1 microgram ml(-1) (24 h) and 0.8 +/- 0.1 microgram ml(-1) (48 h) vs 0.03 +/- 0.02 microgram ml(-1) (9 days) for tenofovir. In contrast, penciclovir and lobucavir lost most of their anti-viral activity when present on the cells for 48 h or less.