Epstein-Barr virus (EBV) infects human B lymphocytes and efficiently transforms them into immortalized lymphoblasts. EBV-determined nuclear antigen (EBNA) and EBV latent infection membrane protein (LMP) are expressed in latently infected, growth-transformed lymphoblasts. To elucidate the functions of EBNA and LMP, clones of cells were established that stably expressed EBNA-1, EBNA-2, EBNA-3A, EBNA-leader protein (EBNA-Lp) or LMP, using gene transfer technique and the growth characteristics of the transfectants were examined. The expression of EBNA-1, EBNA-2,EBNA-3A,EBNA-Lp or LMP caused shortening of doubling time, increased saturation cell density, reduced serum dependence, anchorage-independent growth in semisolid agar and activation of c-myc. Furthermore, the expression of LMP in NIH/3T3 cells led to tumorigenicity in nude mice, enhanced expression of H-ras and increased production of diacylglycerol, which might activate protein kinase C. B cell line, BJAB, EBNA-1 was responsible for expression of c-fgr mRNA and EBNA -2 specifically induced expression of B-cell activation antigens, including CD21 (CR2) and CD23 (Fc epsilon receptor). These results indicate that EBNA and LMP play an important role in EBV-induced growth transformation. It is possible that EBNA-1 and EBNA-2 are directly involved in the early process of immortalization. It is also possible that LMP could contribute to tumorigenic alteration of immortalized cells. The proliferation of the EBNA or LMP-expressing cells was markedly enhanced by phorbol ester. By contrast phorbol ester had no effect on the proliferation of nonexpressing control cells. The phorbol ester enhancement of EBV-induced growth transformation is likely to be mediated by EBNA and LMP.