The involvement of intracellular acidic vesicles in the early phase of Japanese encephalitis (JE) virus infection in Vero cells was observed by adding a specific vacuolar type H⁺-ATPase (V-ATPase) inhibitor (bafilomycin A1) in the cell culture medium. Studies with the detection of viral envelope (E) protein suggested that bafilomycin A1 inhibited virus infection in the cells. Subcellular distribution of incoming biotinylated virions and ³H-uridine-labeled viral RNA were observed in fractions of a Percoll density gradient. At 10min of the chasing period, virions and viral RNA were found mainly in fractions with a mean density of 1.04g/ml corresponding to the endosome both in the control and bafilomycin A1-treated cells. At 60min of the chasing period, the peak of biotin activity was detected in fractions with a mean density of 1.08g/ml corresponding to the lysosome, whereas the peak of radioactivity did not run parallel with that of biotin and shifted to fractions with a mean density of 1.05g/ml and higher than 1.084g/ml, respectively. At 60min of the chasing period in bafilomycin A1-treated cells, the peak of biotin and radioactivity were still found mainly in the fraction with a density of 1.04g/ml, representing the endosome. Subcellular fractionation by a Percoll density gradient revealed that bafilomycin A1 treatment resulted in the accumulation of virions in the endosome fraction and suggested the prevention of intracellular translocation of the virions which occurs during the early entry process of an infecting virus to the cells.