A new standardized test for hepatitis B virus (HBV) DNA with increased sensitivity and range over previous assays (30 to 106 HBV genomes/test) was evaluated in this study. The quantitative results from the test have been validated using international reference specimens of known titer and a reference solution hybridization test. The test has small variability considering the wide dynamic range. The CV was 14% within one experiment and 32% to 39% between independent experiments. Hepatitis B surface antigen (HBsAg)-negative, anti—HBc-positive blood donor sera (n = 25) were all negative for HBV DNA in the new test, whereas 63% (n = 19) of HBsAg-positive healthy carriers had measurable quantities of HBV DNA. In five example cases of chronic hepatitis B patients responding to alfa-interferon treatment but remaining virus positive, HBV DNA was consistently present in posttreatment sera in a titer range 4 × 103 to 106/mL not detectable by the conventional hybridization test. In two complete responders, the HBV DNA titer decreased over six orders of magnitude to below cutoff of the test. In four liver transplant recipients with chronic hepatitis B, viral recurrence was detected by the new test at an early stage much before the clinical relapse. Unlike serology, the test was suitable also in patients under anti-HBs immunoprophylaxis. In conclusion, the new colorimetric polymerase chain reaction (PCR) test allowed thousandfold increased sensitivity in quantification of HBV DNA in patient sera. The test may have future applications in improving assessment of efficacy of antiviral treatment and guiding therapeutic interventions.