The infection of human T cells by Epstein-Barr virus (EBV) may result in a fatal hemophagocytic syndrome (HS). We have previously shown that EBV can selectively upregulate the tumor necrosis factor-alpha (TNFalpha) gene and lead to activation of macrophages in a manner similar to the pathobiology of HS in EBV-infected T lymphoproliferative disorders (LPDs). This study was designed to further clarify the specific EBV gene product(s) responsible for TNFalpha upregulation. RT-PCR analysis of EBV gene expression was performed on 2 CR2-transfected EBV-infected T lymphoma lines and 2 EBV-infected B cell lines. To identify the EBV gene responsible for upregulation of TNFalpha, 2 reporter recombinant plasmids, pTNF-CAT and pTNFalpha-Luc, were then constructed and cotransfected with the expression plasmids of the EBV latent and lytic genes (EBNA-1, EBNA-2, LMP-1, LMP-2A, and BZLF-1) in both T and B cell lines. Analyses using ELISA and Western blotting were further performed to detect the secreted TNFalpha. The results revealed that EBNA-1 and LMP-1 were consistently expressed in EBV-infected T cell lines (type II latency), while a type III latency with expression of EBNA-1, EBNA-2, LMP-1, and lytic BZLF transcripts was detected in EBV-infected B cell lines. LMP-1 was demonstrated to be the only EBV gene product to transactivate the TNFalpha gene, and this phenomenon was observed only in T, not in B, cells. Enhanced secretion of TNF-alpha protein was also detected in LMP1-transfected T cell lines. We concluded that LMP1 is the candidate protein in the upregulation of the TNFalpha gene in T cells and is probably responsible for the pathogenesis of HS in EBV-infected T lymphoproliferative disorders.