Phosphonoacetate was found to be an effective inhibitor of the replication and cytopathic effects (CPE) associated with Anatid herpesvirus (AHV) infections in avian cells. In low multiplicity of infection (MOI) (10(-3) or less) infected Pekin duck and chicken fibroblast cultures, the dosage required for a 50% plaque reduction was approximately 20 microgram/ml. The amount of the inhibitor needed for complete prevention of CPE was found to be MOI dependent with up to 190 microgram/mL required at a MOI of 1.1. Delayed addition of phosphonoacetate to AHV-infected cultures resulted in differing CPE. When added before 25 h postinfection, the CPE were largely prevented. Added between 25 and 40 h postinfection, the CPE consisted of nonproductive, nonperforate focal areas containing viable and nonviable cells. The focal areas did not appreciably increase in size or number in the continued presence of phosphonoacetate, were chromophilic, and tended to be replaced by morphologically normal cells, provided the presence of phosphonoacetate was continued. Maintaining phosphonoacetate in the presence of infected cultures for periods of 7 days or longer resulted in curing and a complete loss of infections virus in the medium and in cell lysates.