The transcriptional template for duck hepatitis B virus (DHBV) replication is believed to be the supercoiled covalently closed circular (CCC) molecule. DHBV CCC DNA can be amplified at least 50-fold in acutely and congenitally infected hepatocyte cultures but is normally maintained at a constant copy number in vivo infections. Here we describe experiments to determine the half-life of DHBV CCC DNA in congenitally infected hepatocyte cultures using both direct and indirect labeling of DHBV CCC DNA with the DNA labeling agent 5-bromo 2-deoxyuridine (BrUdR). Direct labeling of DHBV CCC DNA with BrUdR generated a very stable molecule with no calculable half-life. For indirect labeling experiments, hepatocytes were first cultured for 5 days in the absence of BrUdR to generate a pool of unlabeled DHBV CCC DNA, in then BrUdR was added to the culture medium. By following the fate of the pool of unlabeled CCC DNA in the cultures over time we calculated the half-life of DHBV CCC DNA to be 3 and 5 days in two separate experiments. This result suggests that there is a requirement for continuous amplification of DHBV CCC DNA to maintain a persistent chronic infection.