Lamivudine is the first nucleoside analogue that was shown to have a potent effect on hepatitis B virus (HBV). However, the emergence of mutants resistant or cross-resistant to nucleoside/nucleotide analogues remains a serious problem. Several assays for the detection and quantification of antiviral-resistant mutants have been reported, but it has been difficult to measure the amounts of mutants accurately, especially when the target strain is a minor component of the mixed population. It has been shown that accurate measurement of a minor strain is difficult as long as a matching reaction with a single probe is included in the assay. We developed a new method for the quantification of lamivudine-resistant strains in a mixed-virus population by real-time PCR using minor groove binder probes and peptide nucleic acids, and we achieved a wide and measurable range, from 3 to 10 log 10 copies/ml, and high sensitivity, with a discriminative limit of 0.01% of the predominant strain. The clinical significance of measuring substitutions not only of M204 but also of L180 residues of HBV polymerase was demonstrated by this method. This assay increases the versatility of a sensitive method for the quantification of a single-nucleotide mutation in a heterogeneous population.Chronic liver diseases due to hepatitis B virus (HBV) infection are still serious problems worldwide, and many patients suffer from liver cirrhosis and hepatocellular carcinoma (2, 6, 21). Nucleoside/nucleotide analogues have been introduced in the treatment of chronic hepatitis B, and inhibition of disease progression has been achieved (20). However, since several analogues have been developed and have come into wide use, the risk of the emergence of resistant or cross-resistant mutations is increasing. This is a serious problem, since it may lead to the virological and biochemical breakthroughs that result in the loss of therapeutic effects (18).Lamivudine (3TC) is the first nucleoside analogue that was demonstrated to have a potent effect on HBV (8, 17). The results of many studies on lamivudine-resistant mutants have been reported, and the impact of alteration from M204 to I204 or V204 (YMDD mutations) was initially investigated (20). Thereafter, the clinical significance of other regions of HBV polymerase was clarified. We reported the significance of dual mutations of the YMDD motif and the LLAQ motif (specifically the L180 residue) for the severity of breakthrough hepatitis (29). Recently, alteration of the L180 residue has been noted as one of the most crucial changes for the emergence of entecavir-resistant mutants.It is important to detect mutants as early as possible in order to confirm genotypic or phenotypic resistance that may be followed by virological breakthrough and rebound. Several methods using PCR or hybridization for the qualitative detection of antiviral-resistant mutants have been reported. Some assays have been proposed as sensitive methods for the quantitative measurement of mutant strains (33), and we have reported a quantificat...