To elucidate the mechanism of leukemogenesis induced by bovine leukemia virus (BLV), the abnormality of p53 tumor suppressor gene was examined using the sequencing method of polymerase chain reaction (PCR)-amplified DNA from peripheral blood lymphocytes (PBL) and tumors from BLV-infected cattle that showed evidence of different stages during the progression of enzootic bovine leukosis (EBL). Mutations of the p53 gene were found in tumor cells from cattle with EBL, but not in PBL from BLV-free normal cattle or BLV-infected cattle without any evidence of tumor, suggesting mutation of p53 gene occurred specifically at the lymphoma stage of the disease. Twelve of eighteen cattle with EBL had seven missense and five silent mutations. The mutations were mapped to the highly evolutionarily conserved regions of p53 gene, and were involved in the DNA binding of p53. Thus, it appeared that the p53 point mutation is one of the critical events leading from the asymptomatic stage to the lymphoma stage.