Pyothorax-associated lymphoma (PAL) is a rare tumor associated with long-standing tuberculous pyothorax. Most of these lymphomas are B-cell lymphomas of high-grade malignancy. Over 50 cases have been reported in Japan, but no cases have been described in Western countries. Its pathogenesis remains unknown. We studied immunohistologically the expression of Epstein-Barr virus- (EBV) encoded latent gene products, EB nuclear antigen-2 and LMP-1, in four cases of PAL. Fifty B-cell lymphomas unrelated to pyothorax, and five EBV-bearing lymphoblastic tumors produced in severe combined immune deficient mice (severe combined immune deficient-EBV+ tumors) were also studied as controls. Marked expression of EB nuclear antigen-2 was demonstrated on all four PALs. LMP-1 was also present in all cases, but both the staining intensity and the number of stained cells remained less than on severe combined immune deficient-EBV+ tumors. Neither EB nuclear antigen-2 nor LMP-1 was observed in the 50 control B-cell lymphomas. Additional molecular genetic analysis revealed that EBVs are incorporated into each PAL clonally. These results confirm the definite association of EBV with PALs, although the significance of weak expression of LMP-1 awaits further study.