Association of Epstein-Barr virus (EBV) with T-cell lymphomas was examined by in situ hybridization (ISH) with an antisense probe specific to abundantly expressed EBV-encoded small RNA-1 (EBER1). In addition to EBER1, EBV-specific nuclear antigen-1 (EBNA-1) is commonly expressed in EBV-associated tumors and latently infected B-lymphocytes. We examined paraffin sections of T-cell lymphomas except those of nasal origin for expression of latent viral transcripts by ISH. Using ISH with improved antisense RNA probe specific to EBNA-1 mRNA, the virus was detected in 19 (59%) of 32 cases, whereas the EBER1 transcript was found in only 15 (47%) of 32 cases by conventional EBER-ISH, resulting in 21 EBV-positive cases (66%) by combining the two methods. Latent membrane protein 1 (LMP1) mRNA of EBV was detected in 15 of 32 cases (47%), while no EBNA2 expression was observed in any these tumors. Patients with these lymphomas positive for LMP1 expression showed lower survival rates than those without expression of the viral mRNA. These results indicate that, in addition to EBER-ISH, RNA-RNA ISH with EBNA1 probes could be useful for detection of EBV-infected cells in paraffin sections, and detection of LMP1 mRNA expression in tumor cells could be a useful prognostic factor for T-cell lymphoma.