For quantitative assessment of ongoing symptomatic Epstein-Barr virus (EBV) infection in pediatric recipients of liver transplantation, we determined the number of peripheral blood mononuclear cells (PBMC) infected by EBV by in situ hybridization (ISH) and related the results with clinical courses of those patients. Twenty-four patients had symptomatic EBV infection between February 1995 and March 1996. Blood samples were obtained from these 24 patients at the time of acute phase, from 13 of them during convalescence, and 37 pediatric patients before transplantation. ISH was performed on the PBMC and polymerase chain reaction (PCR) on DNA from whole blood. Oligonucleotide probes for ISH were chosen from coding sequences of EBV-encoded small nuclear RNA 1 (EBER1). Results of ISH were reported in a number of cells expressing EBER1/5 x 104 PBMC (#EBER1). Fever, diarrhea, upper respiratory symptoms, pleural effusion, ascites, lymphadenopathy, and lymphoproliferative disease (LPD) accompanied with EBV infection proven by serology, viral-specific stain or PCR were regarded as EBV related diseases (EBVD). All samples with positive #EBER1 were accompanied by positive EBV PCR. #EBERI was 68.2 +/- 144.9 (mean +/- SD) ranging from 0 to 621 in the acute phase, 0.20 +/- 0.41 ranging from 0 to 2 in the convalescence phase, 0.27 +/- 0.77 in 23 preoperative patients with positive serology, and 0 in all 14 preoperative patients with negative serology. The #EBER1 in ongoing EBVD was significantly greater than that of patients in convalescence or before transplantation. Patients with #EBERI greater than 10 had a significantly lower chance of convalescence and a higher mortality than patients with #EBER 1 less than 10. We conclude that #EBER1 could be a specific and quantitative marker of EBVD and might predict progression to LPD.