Rapid and sensitive methods for the monitoring of influenza virus replication in vitro are needed to address several research questions. Four methods based on different principles were compared: the hemagglutination (HA) assay, the measurement of virus infectivity titers in culture supernatants, the enumeration of infected cells by immunofluorescence and RNA hybridization techniques using digoxigenin (DIG) labeled RNA probes. To this end, MDCK cells were infected at different multiplicities of infection (moi) with a recent influenza A virus (A/Netherlands/18/94 H3N2) and the kinetics of virus replication were monitored with these four assays. At high moi, virus released into the culture supernatant of infected cells was detected by the HA assay 12 h post infection, whereas at lower moi (< or = 0.01) the first HA activity was not detected before 24 h post infection. The measurement of infectious viruses in the culture supernatant proved to be more sensitive, since 4-12 h post infection newly produced virus was detected depending on the moi used. This finding was in agreement with results obtained by the immunofluorescence assay using an antibody preparation specific for the nucleoprotein: single infected cells could be detected as early as 4 h post infection. At this time point, positive signals were also obtained when mRNA/cRNA specific hybridization was carried out for the NP gene segment, but not for viral NP RNA or RNA specific for the hemagglutinin, which were only detected at later time points after infection. Thus, besides direct measurement of infectious virus and immunofluorescence, RNA hybridization proved to be a sensitive assay for monitoring influenza virus replication in vitro.