Longstanding inflammation in chronic pyothorax (CP) plays a role in the development of pleural lymphoma; therefore, in 1987, the term pyothorax-associated lymphoma (PAL) was proposed. A recent study showed in the tumor cells of a limited number of patients, the presence of the Epstein-Barr virus (EBV) genome together with the expression of latent infection genes in the tumor cells of PAL. The purpose of the present study is to evaluate an association of EBV with PAL in a large number of patients. In addition, the presence of the EBV genome was examined in cases with CP without PAL. Histologic, immunohistochemical studies, and analyses by polymerase chain reaction (PCR) and in situ hybridization method for EBV were performed on 34 PAL and 16 CP collected by nationwide study. Median age of the patients in both diseases was approximately 70 years with a marked preponderance in men. Mean duration of CP in patients with CP alone and with CP complicated with PAL was 33 and 37 years, respectively. Histologically all PAL were non-Hodgkin's lymphoma, with immunoblastic type being the most common. Immunohistochemistry revealed 28 of 34 PAL to be B-cell type. Combined PCR, in situ hybridization method, and immunohistochemistry showed that the EBV genome was detected in lymphoma cells in 85% of PAL with almost constant expression of latent membrane protein-1. The EBV genome was detected by PCR in only one of 16 CP.(ABSTRACT TRUNCATED AT 250 WORDS)