We made some improvements on the in situ hybridization protocol for EB virus (EBV) detection so that many general laboratories in Japan are able to perform the procedure. Using FITC-labeled EBER-PNA probes (DAKO), we examined dilutions of several chemicals and the incubation temperature of the probes in tissue sections. We confirmed the accuracy and reproducibility of our protocol by applying PCR to detect EBV genomes within tissue sections. We applied the technique to 55 laboratory samples which were suspected to be infected with EBV, and eight negative control and successfully ended up with pathological diagnosis. Moreover, coupled with immunohistochemical detection of LMP-1, we could determine the type of EBV infection.