Viral hemorrhagic septicemia virus (VHSV) and infectious pancreatic necrosis virus (IPNV) are economically important pathogens of the salmonid aquaculture industry. In previous work we demonstrated that a cell line persistently infected with IPNV (EPCIPNV) exhibited antiviral activity against superinfection with the heterologous virus VHSV. This work extends our study by analyzing the replication of VHSV in the IPNV-persistently infected cells. At early and late stages of infection VHSV RNA synthesis, as well as VHSV-induced syncytia formation, were examined in EPCIPNV cultures. During the course of VHSV infection the accumulation of VHSV RNA is inhibited in EPCIPNV cells. Typical VHSV-induced membrane fusion at the late stages of infection is also absent in the IPNV carrier cultures. VHSV binding and fusion to EPCIPNV cells did not appear to be impaired, but a potent inhibitory effect on VHSV RNA synthesis is exerted at early times of infection in the IPNV carrier culture. In conclusion, the EPCIPNV cells are considered to be a useful system to study viral interference as well to analyze the mechanisms underlying the phenomenon of superinfection exclusion.