Diagnostic assays allowing the quantification of hepatitis B virus (HBV) DNA over a wide range of concentrations are important for monitoring patients during antiviral therapy. The aim of this study was to develop a new real-time method for HBV DNA quantification. Primers and probe were selected in a highly conserved region of the HBV S gene, and a plasmid containing the pre-S/S region was used as a standard. Linear quantification of the standard was obtained between 10 and 10(9) copies/reaction, with high correlation between ayw and adw genomes (P<0.001). HBV DNA was detected in serial dilutions of a high-titer serum sample with linear results until 2.4 x 10(3) copies/ml. One hundred eight serum samples positive for hepatitis B surface antigen were tested in both the real-time assay and the Digene Hybrid Capture assay (Digene, USA). HBV DNA could be detected by both assays in 70 samples, with significant correlation of results (P<0.001). Results for 38 samples were below the sensitivity limit of the Digene assay, but they could be quantified by the real time polymerase chain reaction assay. These results show that real-time polymerase chain reaction allows sensitive, rapid and linear quantification of HBV DNA in serum.