Three woodchucks infected persistently with the woodchuck hepatitis virus (WHV) were treated with acyclovir (ACV) to investigate the effect of inhibiting viral DNA synthesis upon the replication of an orthohepadnavirus in vivo. Normal viraemia was reduced during the treatment period in all three animals, but each responded with a distinct serum phenotype. In the most provocative case, the profile of the WHV DNAs in both the liver and serum provided a simple and novel description of the orthohepadnaviral infection for this ACV protocol. The pre-drug viraemia was rapidly cleared from the serum and replaced by virion-like particles containing predominantly minus-strand WHV DNAs. These serum DNA species had the character of replicative intermediates arrested in their elongation by ACV-mediated chain termination and were contained in particles with a buoyant density in CsCl essentially identical with virions. However, in infected hepatocytes, initiation of reverse transcription within newly formed core particles was not inhibited by the ACV treatment. Instead, an heterogeneous array of minus-strand DNAs were synthesised, each presumed to be truncated by the incorporation of one molecule of ACV monophosphate. An approximately normal level of core particles was present in the liver of this woodchuck after 26 days of the ACV protocol; excess drug-arrested nucleocapsids were steadily removed throughout the dosing period upon their envelopment and secretion as virion-like particles into the circulation. These data suggest that plus-strand DNA synthesis may not be absolutely required prior to secretion of virus from the infected cell. J Med Virol 51:6–16, 1997. © 1997 Wiley-Liss, Inc.