The in vitro and in vivo antiviral activities of two mononucleoside phosphotriester derivatives of acyclovir (ACV) incorporating S-acyl-2-thioethyl (SATE) groups are reported using the duck model of hepatitis B (DHBV). In primary duck hepatocyte cultures, the described phosphotriesters significantly inhibited the replication of DHBV at submicromolar concentrations. They were found to be more potent than the parent nucleoside. This result was in agreement with our data concerning the anti-HBV activity of these pronucleotides in HepG2.2.15 cells (previous paper). In vivo, the studied SATE pronucleotide was also found to be more efficient than ACV in infected ducklings upon short-term oral therapy, while intraperitoneal treatment showed high anti-DHBV activity with both ACV and its SATE pronucleotide in this animal model. These findings demonstrate the potential of SATE pronucleotides of ACV as anti-HBV agents.