We evaluated the safety and efficacy of long-term lamivudine monotherapy in a group of 25 patients with hepatitis B e antigen (HBeAg)-negative chronic hepatitis B. Lamivudine was administered in a daily dose of 150 mg for a mean of 26 ± 7 months and was well tolerated. No patient lost hepatitis B surface antigen (HBsAg). The rate of initial biochemical response increased from 88% at 6 months to 96% at 12 months of therapy, but it progressively decreased thereafter; the biochemical remission rate was 68% at 18 months, 59.5% at 24 months, and 42.5% at ≥30 months. Alanine transaminase (ALT) increased to higher than the baseline levels in 8 of the 11 patients with a biochemical breakthrough reaching acute hepatitis levels in 6 of them. Acute icteric hepatitis developed in one patient. The virologic remission rate assessed by a sensitive quantitative polymerase chain reaction (PCR) assay was 68% at both 6 and 12 months, decreasing thereafter to 52% at 18 months and to 41.6% at both 24 and ≥30 months. Virologic breakthroughs were always persistent and preceded ALT elevations by a median of 4 (3-24) months. YMDD mutants were detected in all patients with a virologic breakthrough. In conclusion, in patients with HBeAg-negative chronic hepatitis B, long-term lamivudine therapy is safe and is associated with high biochemical and virologic response rates at the end of the first year. However, response rates tend to decrease with time and breakthroughs due to YMDD mutants accumulate. ALT activity during breakthroughs often exceeds the baseline and may reach even acute hepatitis levels.