Numerous studies have shown that resistance to long-term lamivudine therapy occurs in as many as ⅔ of hepatitis B virus (HBV) chronic carriers. Additional studies have shown that reversion of HBV mutations in the precore/core promoter region conferring an HBeAg-negative phenotype/genotype can occur in up to 30% of lamivudine-treated patients. In this study, sequences of the HBV polymerase and precore/core coding regions in 26 HBV-infected patients (24 with HBeAg-negative virus infection, 25 genotype D, 1 genotype A) treated for 27 to 53 months with lamivudine were analyzed to determine the relationship between pretreatment HBV DNA sequence patterns and long-term treatment response, and the effect of therapy on the status of HBV precore mutations. Reversions of precore mutations A1762T/G1764A and G1896A were observed in 29% and 25% of patients, respectively, but none became HBeAg-positive. These data are consistent with previously published reversion frequencies for 2 other groups of lamivudine-treated patients. Two naturally-occurring DNA polymorphisms at aa91 and aa256 of the HBV polymerase were observed in the pretreatment serum samples, which correlated with extended treatment failure. In conclusion, reversion of mutations conferring an HBeAg-negative phenotype occur relatively frequently under lamivudine therapy. Furthermore, at least in HBeAg-negative patients infected predominately with HBV genotype D, specific viral DNA sequences which are present before therapy appear to be useful as predictors of long-term response to lamivudine treatment. (HEPATOLOGY 2004;39:64–73.)