A herpesvirus (RhEBV) was isolated from a lympho‐blastoid cell line (LCL) that became established from a malignant lymphoma in a rhesus monkey. The predominant cell marker in the LCL was that of B lymphocytes. RhEBV‐induced viral capsid (VCA) and nuclear antigens (NA) in the LCL were serologically related to similar antigens known to be induced by human Epstein‐Barr virus (EBV). RhEBV was of non‐human primate origin and was clearly differentiated from EBV in the anti‐complement immunofluorescence reaction using human and non‐human primate sera with antibodies to the NA induced by the respective viruses. While human sera reacted with NA induced by both EBV and RhEBV, monkey sera failed to recognize the NA induced by EBV. RhEBV‐induced NA was present in nearly all the cells of a suspension prepared from the tumor tissue mass, but not in the monolayer fibroblasts derived from the tumor tissue or in the blood and lymph‐node lymphocytes of clinically healthy animals. RhEBV induced in vitro transformation and establishment of LCLs from peripheral blood lymphocytes of normal rhesus and cynomolgus monkeys but not from those of 6 other non‐human primate species tested. The LCLs, with predominant B‐lymphocyte markers, established after treatment with RhEBV, all had evidence of the virus infection since nearly all cells in the culture expressed the virus‐induced NA.