We have reported the prevalence of a fulminant hemophagocytic syndrome (HS) in previously healthy young children in Taiwan, most of which probably represent a lethal form of primary or active Epstein-Barr virus (EBV) infection. To further confirm their EBV association, in situ EBV hybridization (ISH) was performed on tissue biopsies from 15 pediatric HS patients (median age, 3 years and 4 months) using digoxigenin-labeled RNA probes EBER1. Double labeling immunostaining and ISH was then performed to define the immunophenotype of the lymphoid cells containing the EBV transcripts. Among the 13 patients who had serological evidence of acute or active EBV infection, 9 had demonstrable EBER1 transcripts in bone marrow, liver, and/or skin biopsies. EBER1-specific signal was not detectable in the two specimens from EBV-seronegative patients. The distribution of EBV-containing cells could be extensive or scattered. To our surprise, the EBER1 transcripts existed exclusively in T lymphoid cells in all nine cases examined rather than in B cells as previously believed in infectious mononucleosis. Considering the young affected age of the HS patients and the serological response to EBV, we suggest that EBV can infect T cells in primary EBV infection and the proliferation of these EBV-infected T cells may be responsible for the ominous outcome in childhood HS patients in Taiwan.