The virion half-life of hepatitis B virus (HBV) is currently estimated at approximately 1 day. This estimate has been obtained from drug perturbation experiments with reverse transcriptase inhibitors. However, the analyses of those experiments have not considered the export of virions produced from preformed mature DNA-containing HBV capsids in infected cells. Data from 3 acutely infected chimpanzees indicates that there is approximately 10-fold more total intracellular HBV DNA than HBV DNA in blood, and therefore the half-life of virions for chimpanzees during acute infection is 10-fold shorter at 3.8 hours than the half-life associated with export of total intracellular HBV DNA. Mathematical model simulations duplicating the viral dynamics observed in drug perturbation experiments suggest a half-life of at most 4.4 hours for HBV virions in chronically infected humans, significantly shorter than current estimates, but consistent with the half-lives of virions for hepatitis C virus and HIV. This faster turnover of HBV in blood indicates a correspondingly higher replication rate and risk of mutation against hepatitis B antiviral therapy. In conclusion, we find the half-life of HBV virions is approximately 4 hours, significantly shorter than current estimates of 1 day. This new value is consistent with virion half-life estimates for HIV and hepatitis C virus. (HEPATOLOGY 2006;44:1117–1121.)