Our previous studies found that patients with B-cell non-Hodgkin lymphoma (NHL) had a higher incidence of hepatitis B virus (HBV) infection in serum than patients with T-cell NHL or other cancers. We sought to identify a possible role of HBV infection in B-cell NHL tumorigenesis and to understand its underlying clinical relevance. Fresh and paraffin-embedded primary tumor tissue from patients with NHL as well as from those with other lymphatic system diseases were investigated by PCR and immunohistochemistry. Many more patients with B-cell lymphoma whose serum was positive for hepatitis B surface antigen (HBsAg) were also positive for HBV-DNA than were those with T-cell NHL or other lymphatic system diseases whose serum was positive for HBsAg, in both fresh (55 vs. 15.4%) and paraffin-embedded (38.3 vs. 11.8%) tissue. Positive expression of the HBV-associated proteins HBsAg and hepatitis B core antigen was found in B-cell NHL lymphocytes and endothelial cells. Only 8.3% of patients with B-cell NHL who were negative for HBsAg but positive for other HBV markers were positive for HBV-DNA in tumor tissue. These results suggest that chronic HBV infection in lymph nodes could be associated with B-cell lymphoma.