Success in antiviral therapy for chronic hepatitis B is supported by highly sensitive PCR-based assays for hepatitis B virus (HBV) DNA. Nucleic acid extraction from biologic specimens is technically demanding, and reliable PCR results depend on it. The performances of the fully automatic system COBAS AmpliPrep-COBAS TaqMan 48 (CAP-CTM; Roche, Branchburg, NJ) for HBV DNA extraction and real-time PCR quantification were assessed and compared to the endpoint PCR COBAS AMPLICOR HBV monitor (CAHBM; Roche). Analytical evaluation with a proficiency panel showed that CAP-CTM quantitated HBV DNA levels in one single run over a wide dynamic range (7 logs) with a close correlation between expected and observed values (r = 0.976, interassay variability below 5%). Clinical evaluation, as tested with samples from 92 HBsAg-positive patients, demonstrated excellent correlation with CAHBM (r = 0.966, mean difference in quantitation = 0.36 log(10) IU/ml). CAP-CTM detected 10% more viremic patients and longer periods of residual viremia in those on therapy. In lamivudine (LAM)-resistant patients, the reduction of HBV DNA after 12 months of Adefovir (ADF) was higher in the combination (LAM+ADF) schedule than in ADF monotherapy (5.1 logs versus 3.5 logs), suggesting a benefit in continuing LAM. CAP-CTM detected HBV DNA in liver biopsy samples from 15% of HBsAg-negative, anti-HBcAg-positive graft donors with no HBV DNA in plasma. The amount of intrahepatic HBV DNA was significantly lower in occult HBV infection than in overt disease. CAP-CTM can improve the management of HBV infection and the assessment of antiviral therapy and drug resistance, supporting further insights in the emerging area of occult HBV infection.