Persistence and diuretic shedding of hepatitis B virus (HBV) by mosquitoes (Diptera: Culicidae) was studied by using infectious blood feedings, intrathoracic inoculations, and detection of virus by polymerase chain reaction (PCR) and Southern hybridization. Results showed that both Anopheles stephensi Liston and Ochlerotatus triseriatus (Say) shed HBV during diuresis for up to 72 h after feeding on an HBV-positive serum drawn from a human donor. HBV did not persist in the bodies of either An. stephensi or Oc. triseriatus past 72 h by infectious feeding or intrathoracic inoculation of HBV suspension. Viral dissemination did not occur by infectious feeding in An. stephensi or Oc. triseriatus, or by intrathoracic inoculation in An. stephensi, Oc. triseriatus, or Culex quinquefasciatus Say. These results suggest that HBV could be transmitted to humans by a stercorarian route, especially if mosquitoes that fed on an HBV-positive human are interrupted during feeding and move to another person to resume feeding.