(2-Phosphonylmethoxyethyl) adenine (PMEA) was evaluated for its inhibitory effect on hepadnavirus replication in three different cell systems, ie, human hepatoma cell lines HepG2 2.2. 15 and HB611 (transfected with human hepatitis B virus (HBV)) and primary cultures of duck hepatocytes infected with duck hepatitis B virus (DHBV). PMEA inhibited HBV release from HepG2 2.2. 15 cells and HB611 cells at a 50% inhibitory concentration (IC 50) of 0.7 and 1.2 μM, respectively. Intracellular viral DNA synthesis was inhibited at concentrations equivalent to those required to inhibit virus release from the cells. DHBV secretion from duck hepatocytes was inhibited by PMEA at an IC 50 of 0.2 μM. HBsAg secretion was inhibited by PMEA in a concentration-dependent manner in HB611 cells and DHBV-infected duck hepatocytes but not HepG2 2.2. 15 cells. The 50% cytotoxic concentration, as measured by inhibition of [3 H-methyl] deoxythymidine incorporation was 150 μM for the two human hepatoma cell lines and 40 μM for the duck hepatocyte cultures. In a pilot experiment PMEA was found to reduce the amounts of DHBV DNA in the serum of Pekin ducks.