Simian virus 40 (SV40) is associated with some systemic non-Hodgkin's lymphomas (NHL) among HIV-positive patients, based on assays for viral DNA sequences. To investigate the possible production of the viral transforming protein, we examined age-matched case-control specimens from patients with HIV/AIDS for the expression of SV40 large tumor antigen (T-ag). Masked specimens initially examined by polymerase chain reaction (PCR) for polyomavirus and herpesvirus DNA sequences were assessed for the expression of SV40 T-ag and phenotypic lymphocyte markers by immunohistochemistry (IHC). Fifty-five systemic NHL and 25 nonmalignant lymphoid and malignant nonlymphoid tissue control cases from two HIV community programs in Texas and New Jersey were scored for IHC positivity without knowledge of the PCR results. IHC showed expression of SV40 T-ag among B-cell lymphomas, whereas none of the control tissue samples were positive for T-ag (12/55, 22% vs. 0/25, 0%; P = 0.01). SV40 T-ag expression was detected only in B-cell lymphoma specimens that contained SV40 DNA sequences. Not all lymphoma cells in a positive specimen stained for T-ag, and the reaction was lower intensity than observed in SV40 hamster tumors. SV40 T-ag was demonstrated in both primary and recurrent tumors from one patient. A germinal center B-cell-like (GCB) profile was more frequently expressed by SV40-positive tumors than in Epstein-Barr virus (EBV)-related lymphomas (10/12, 83% vs. 6/13, 46%; P = 0.05), whereas a non-GCB phenotype was more frequent in EBV-positive than in SV40-positive lymphomas (7/13, 54% vs. 2/12, 17%; P = 0.05). This study shows that SV40 gene expression occurs in a fraction of cells in some B-cell lymphomas among patients with HIV/AIDS.