Pyothorax-associated lymphoma (PAL) is a B cell lymphoma that develops in Japanese patients with tuberculosis-associated chronic pyothorax (TaCP). Epstein-Barr virus (EBV) has been shown to be causally related to PAL. To clarify the developmental process of PAL, the systemic and local presence of EBV, and serum profile of anti-EBV antibodies was investigated in TaCP. EBV genome was found in peripheral blood mononuclear cells by PCR in a 10(-4)-10(-5) amount of Raji cell-DNA in three of four patients with TaCP, but was also identified in patients with pyothorax caused by other diseases (2/2) or without pulmonary diseases (2/6). EBER1 in situ hybridization and EBNA2 immunocytochemistry revealed clusters of EBV-carrying cells in the cavity content (3/18) but not at the pyothorax wall; EBV(+) histological lymphoma cells were found in two cases and EBV(+) mononuclear cells were found in one case. A simultaneous increase in serum titers of anti-EBV viral capsid antigen IgG and IgA antibodies was observed in TaCP (4/16). These results suggest that a local factor, an inflammatory cavity, has a pivotal role in the development of PAL, which might be reflected in the serum titers of anti-EBV antibodies in patients with TaCP.