We have shown previously that cancer/testis (CT) antigen, CT45, is expressed in various epithelial cancers at a frequency of <5% to approximately 35%. In this study, the protein expression of CT45 was examined in non-Hodgkin B-cell lymphomas and classical Hodgkin lymphoma by immunohistochemical analysis. Serological response to CT45 was also evaluated by ELISA using CT45 recombinant protein and sera from patients with Hodgkin lymphoma. None of the 80 low-grade B-cell lymphomas, including chronic lymphocytic leukemia/small lymphocytic lymphoma, follicular lymphoma, and mantle cell lymphoma, expressed CT45. In comparison, CT45 was expressed in 28 of 126 (22%) diffuse large B-cell lymphomas (DLBCL). A remarkably high percentage (42/72, 58%) of classical Hodgkin lymphoma contained CT45-positive Reed-Sternberg cells. Nodular sclerosis and mixed-cellularity subtypes had similar frequency of CT45 expression, but most EBV-positive cases were CT45 negative. Gray-zone lymphoma (cases with features of both DLBCL and classical Hodgkin lymphoma) also showed frequent (64%) CT45 expression. Evaluation of reactive lymphoid tissues showed scattered CT45-positive lymphocytes in a single case of florid follicular hyperplasia, raising the possibility that this case was an evolving malignancy. Despite frequent CT45 expression, only 1 of 67 Hodgkin lymphoma patients had detectable anti-CT45 antibodies in the serum, suggesting that the immune response to CT45 may be suppressed. In conclusion, classical Hodgkin lymphoma has the highest frequency of CT45 expression among all malignancies tested to date, the frequency of CT45 expression in DLBCL is similar to that seen in epithelial cancers, and low-grade non-Hodgkin B-cell lymphomas do not express CT45.