A previous study has shown that the BNLF1 of Epstein-Barr virus (EBV), isolated from a nasopharyngeal carcinoma biopsy (BNLF1-1510), was able to transform Balb/3T3 cells. On the other hand, BNLF1 of a prototype virus B95-8 (BNLF1-958) was not transforming unless the gene was transcribed from a strong promoter. In this study, we have generated chimeric BNLF1 by exchanging the DNA fragments between BNLF1-1510 and BNLF1-958 and examined their expression and transformation ability in Balb/3T3 cells. Results showed that transformation of Balb/3T3 cells by BNLF1-1510 was not due to the excessive expression of the gene. Transfection of Balb/3T3 cells with chimeric BNLF1 showed that the genes with 3' 453 bp sequence of BNLF1-1510 were oncogenic to the cells. Study also revealed that changing the numbers of the 33 bp repeats in the 3' region of the two BNLF1s did not affect the transformation characteristics. On the other hand, deletion of a 30 bp sequence of BNLF1-958, which is absent in BNLF1-1510, changed the gene from non-oncogenic to oncogenic and insertion of this 30 bp sequence into BNLF1-1510 abolished the transformation ability. BNLF1 without this 30 bp sequence was also found in the tumours of other EBV-related neoplastic disease, suggesting that absence of this 30 bp sequence in BNLF1 may be associated with the oncogenesis of these diseases.