When mouse-adapted influenza virus A/PR/8/34 (A/PR8) (10 PFU/cell) was adsorbed to Madin-Darby canine kidney (MDCK) cells at 4°C for 1 h and incubated at 37°C, release of the virus from the cells was detected in the medium from 4 h after incubation and reached to plateau at 8 h. However, 5,7,4 -trihydroxy-8-methoxyflavone (F36) from the roots of Scutellaria baicalensis significantly reduced this single-cycle replication of A/PR8 from 4 h to 12 h after incubation by dose-dependent manner and the dose which decrease the virus titer one tenth was 11 μM. F36 (50 μM) did not inhibit the adsorption of A/PR8 to MDCK cells, but reduced release of the virus in the medium, when it was added at 0 or 2 h after the incubation. The cell-associated virus determined by sialidase activity was also reduced by F36 treatment at 0 or 2 h. F36 also inhibited the fusion of A/PR8 with liposomes containing bovine brain mixed gangliosides at pH 5.0. However, F36 little affected on the elongation activity of the viral RNA-dependent RNA polymerase in vitro. These results suggest that F36 reduces the replication of A/PR8 by inhibiting the fusion of the virus with endosome/lysosome membrane which occurs at early stage of virus infection cycle. Whereas, when F36 was added to the MDCK cells infected with A/PR8 at 3 or 4 h after incubation, release of the virus in the medium was reduced but the cell-associated virus was increased in comparison with control. Scanning and transmission immunoelectron microscopic studies revealed that F36 inhibited the budding of progeny A/PR8 from the MDCK cell surface and microvilli, when it was added at 3 h after incubation. The accumulation of the A/PR8 antigen was observed on the cell surface by immunofluorescence and transmission immunoelectron microscopies by the addition of F36. These results suggest that F36 also shows anti-influenza virus activity against A/PR8 by inhibiting the budding of the progeny virus from the cell surface, when it was added at budding stage of virus infection cycle.