Malignant lymphomas, which were usually of T-cell type, were induced in 10 of 13 (77%) male rabbits (Japanese white, 8/10; New Zealand white, 2/3) inoculated i.v. with HTLV-II-transformed simian (Cynomolgus) leukocyte cell line (Si-IIA) cells. Of 7 rabbits injected with cell-free pellets from Si-IIA cultures, 5 also developed malignant lymphoma (15-28 days). Lymphoma development was completely inhibited by inactivation of cell-free pellets from Si-IIA culture with ethyl ether and was almost suppressed by neutralization of the cell-free pellets with anti-Si-IIA sera. Herpesvirus particles were discovered very rarely in Si-IIA cells, in addition to C-type virus particles, by electron microscopy. Si-IIA cells were positive for Epstein-Barr-virus (EBV)-associated nuclear antigen (EBNA) by immunofluorescence (IF) test. Antibody response to viral capsid antigen of EBV was also detected in sera from rabbits inoculated with Si-IIA. EBV-encoded RNA-1 (EBER-1) was demonstrated in Si-IIA, the tumor tissues and all rabbit tumor cell lines by in situ hybridization. EBV DNA was also detected in Si-IIA and rabbit lymphoma cell lines by polymerase chain reaction (PCR) and Southern blotting. However, EBV DNA was amplified only by some primers complementary to human EBV sequence (B95-8), but not by other primers. Integration of HTLV-II provirus genome could not be detected in Si-IIA-induced rabbit tumor cells. Moreover, no lymphoma was induced by inoculation of HTLV-IIC and MOT (other HTLV-II-producing human cell lines), B95-8(EBV-producing cell line) or TALL-1 and peripheral leukocytes from normal Cynomolgus (controls). Neither Herpesvirus saimiri nor H. ateles (simian oncogenic viruses) were detected in Si-IIA cells by IF test. These data suggest that the high rate of lymphoma induction in rabbits may not be caused by HTLV-II, human EBV (B95-8) or well-known simian oncogenic viruses, but by EBV-related herpesvirus derived from Si-IIA cells or HTLV-IIA cells, with which Si-IIA was established. The availability of this animal model promises to clarify the role of EBV in human lymphoma and provides a means of studying prophylactic and therapeutic regimens.