There is no standard therapy for patients with anti-HBe-positive chronic hepatitis B. The aims of this study were to analyse the efficacy of lamivudine therapy for two years in these patients and to study the sequence variations in the precore and polymerase hepatitis B virus (HBV) regions in relation to therapy. Sixteen patients with chronic anti-HBe-positive hepatitis were treated with lamivudine (100 mg) once daily for 2 years. Levels of alanine aminotransferase (ALT), HBV-DNA and HBsAg were monitored during therapy. The polymerase and precore genes were amplified by polymerase chain reaction and their products were sequenced directly. Thirteen of 16 patients (81%) had a virological and biochemical response after 1 year of therapy and 11 (69%) maintained the complete response after 2 years of lamivudine therapy. Among the three patients without initial virological or biochemical response at year 1, prolonging therapy to 2 years was not associated with an increase in the response. YMDD variants were detected in 19% of cases in the first year and in 44% in the second year: YVDD being the most frequent mutations detected during year 1 and YIDD during year 2 of therapy. YMDD variants were found in 7–27% of cases with complete response depending on the duration of therapy. Our results show that prolonging lamivudine therapy is safe, well tolerated and maintains viral inhibition in anti-HBe-positive patients. However, its efficacy tends to decrease overtime and it is associated with an increase in YMDD variants, even in some cases, of complete response.