We describe a procedure for in situ hybridization using a biotinylated Epstein-Barr virus (EBV) sequence with detection at the light and electron microscopic levels. In situ hybridization using an immunogold-silver staining detection system was used to identify biotinylated DNA probes in cell smears and in Lowicryl K4M-embedded EBV-infected and -noninfected cell lines. At the light microscopic level, the reaction product of hybridized EBV DNA sequence seemed to be located mainly in the nuclei. The labelling was dependent on the cell strains. However, at the electron microscopic level, the reaction product was evident as spots or clusters distributed not only in the nuclei of EBV-infected cells but also in the cytoplasm and extracellular particles. These findings suggest that immature particles in the cytoplasm contain EBV DNA. This procedure can be applied to the observation and identification of virus infection.