Malignant lymphoma (ML) induction in rabbits by Epstein-Barr virus (EBV)-related herpesvirus of cynomolgus (Cyno-EBV) is reported. Twenty-seven of 30 (90%) rabbits inoculated intravenously with Cyno-EBV-producing simian (cynomolgus) lymphocyte cell line (Ts-B6) cells developed ML between 45 and 115 days after inoculation. The peroral inoculation of Ts-B6 cells induced ML in only 2 of 10 (20%) rabbits (75 to 85 days). Five of 6 (83%) rabbits injected with cell-free pellets from Ts-B6 cultures also developed ML (27 to 122 days). Antibody response to the viral capsid antigen of EBV was also detected in sera from rabbits inoculated with Ts-B6. ML of the large cell or mixed type infiltrated diffusely in many organs, frequently involving the spleen, liver, kidneys, heart, and less frequently the lungs, lymph nodes, brain, eyes, gastrointestinal tract, thymus, and bone marrow. A chromosomal analysis of five lymphoma cell lines established from tumor-bearing rabbits revealed the rabbit karyotype. Three of these cell lines showed the chromosomal abnormalities with 12q- or t (7p+:12q-). EBV-encoded small RNA-1 and EBV-associated nuclear antigen 1 were expressed in Ts-B6 cells, the tumor tissues, and all rabbit cell lines by in situ hybridization and by immunofluorescence tests, respectively. EBV DNA was also detected in Ts-B6 cells and rabbit lymphoma cell lines by polymerase chain reaction and Southern blot analysis. The Southern blots of EBV termini revealed oligoclonal bands in the Cyno-EBV-induced rabbit lymphomas. No lymphoma was induced by the inoculation of B95-8 (EBV-producing cells) or peripheral leukocytes from normal cynomolgus (controls). These data suggest that the high rate of lymphoma induction in rabbits may be caused not by human EBV (B95-8) but by Cyno-EBV from Ts-B6 cells. A sequence analysis of the IR1 (BamHIW) region of Cyno-EBV revealed that this region is quite similar to that of herpesvirus Macaca fascicularis 1,which is a causative agent for a monkey model of AIDS-retated lymphomas. The present rabbit model of lymphoma with specific chromosomal abnormalities is very useful to clarify the role of EBV in human EBV-associated lymphoma and provides a means for studying prophylactic and therapeutic regimens.