Hepatitis B e antigen (HBeAg)-negative chronic hepatitis B is difficult to treat and there is little long-term data for lamivudine treatment of severe acute exacerbation. We report a prospective, consecutive cohort of severe acute exacerbation of HBeAg-negative chronic hepatitis B patients treated by lamivudine between 1999 and 2004. All patients had respectively increased alanine aminotransferase and serum bilirubin to at least 10 and 2 times the upper limit of laboratory normal. Thirty-two patients were treated with lamivudine for 130 +/- 58 (range 48-217) weeks. Five patients had evidence of virological breakthrough (HBV DNA >10,000 copies/ml) during lamivudine treatment. The cumulative probability of maintained response without any virological breakthrough was 94% (95% confidence interval [CI], 86-100%) at year 1, 94% (95% CI, 82-100%) at year 2 and 71% (95% CI, 41-100%) at year 3. At the last follow-up visit, 31 (97%) lamivudine patients had HBV DNA <10,000 copies/ml. The prevalence of lamivudine resistance mutation was 1 in 32 patients (3%; 95% CI, 0-9%) at year 1, 1 in 17 patients (6%; 95% CI, 0-17%) at year 2, 1 in 9 patients (11%, 95% CI, 0-32%) at year 3 and 1 in 4 patients (25%; 95% CI, 0-67%) at year 4 of lamivudine treatment. In conclusion, extended lamivudine treatment is associated with a high maintained virological response and a low rate of drug resistance in severe acute exacerbation of HBeAg-negative chronic hepatitis B.