Prolonged therapy for chronic hepatitis B (HBV) with nucleoside analogs may result in the emergence of HBV mutants resistant to antivirals. Here, we describe the transient selection of an HBV polymerase gene mutant that was associated with viral persistence in an immune competent patient treated with famciclovir. Viral polymerase gene sequence was analyzed directly on polymerase chain reaction (PCR) products and also after cloning. The results showed the transient selection of a V542I mutant in the C domain of the viral polymerase. This mutation was associated with a stop codon at amino acid position 199 in the overlapping S gene. The mutated sequence was subcloned in a vector expressing the entire HBV pregenome to study its replication capacity after transient transfection in cultured hepatoma cells. The results showed that the V542I mutant has a decreased replication capacity compared with wild type virus and does not produce HBsAg. The sensitivity of the V542I mutant to penciclovir, the active metabolite of famciclovir, was further studied in tissue culture. This mutant was shown to be resistant to penciclovir, but remained sensitive to lamivudine, as was subsequently observed in vivo. These findings indicate that a prolonged administration of famciclovir may allow for the selection of HBV polymerase gene mutants in immune competent patients. The impaired replication capacity of this V542I mutant may have contributed to the absence of outgrowth of this viral strain in vivo. The study of the in vitro sensitivity of HBV polymerase mutants to nucleoside analogs will be important to design new anti-HBV strategies.