Biopsy samples obtained from 20 patients with human immunodeficiency virus (HIV)-associated non-Hodgkin lymphoma (NHL) were assessed for evidence of Epstein-Barr virus (EBV) and HIV sequences. DNA was extracted from formalin-fixed, paraffin-embedded NHL tissue and specific viral gene sequences were sought using the polymerase chain reaction (PCR). EBV sequences were found in 10 NHL samples (50%), with five tumors showing A-type and five B-type sequences. By serologic testing, 18 of 19 patients had antibodies to EBV, with 14 patients having antibodies to A-type EBV and 11 to B-type EBV. Serology confirmed the high prevalence of type B EBV in HIV-infected patients, but was not a reliable indicator of the EBV subtype present in the lymphomas. HIV sequences were present in biopsy tissue but at a level consistent with an origin from bystander HIV-infected cells. All 20 patients were negative by enzyme-linked immunosorbent assay for antibodies to human T-cell leukemia virus-type I. The high prevalence of type B EBV in these tumors is similar to the findings in endemic Burkitt's lymphoma, where 40% of the tumors have type B viral sequences. In normal populations, type B EBV is rarely found outside the nasopharynx. These studies support the hypothesis that EBV is an important cofactor in NHL in HIV-infected persons. The finding that B-type EBV is present in 25% of HIV-associated NHL suggests that this EBV subtype may be an important human pathogen with a wider geographic distribution than originally thought.