We demonstrate that the expression of TRAF1 and activated c-Rel, two proteins that function in signaling events downstream of activated CD30 in Reed-Sternberg cells, reliably distinguish classical Hodgkin lymphoma from anaplastic large cell lymphoma, nodular lymphocyte predominant Hodgkin lymphoma, and nonmediastinal diffuse large B-cell lymphoma. By immunohistochemistry, we found strong TRAF1 staining in 21 of 25 cases of classical Hodgkin lymphoma. In contrast, strong TRAF1 staining was present in only 1 of 17 cases of anaplastic large cell lymphoma, 0 of 15 cases of lymphocyte predominant Hodgkin lymphoma, and 2 of 36 cases of nonmediastinal diffuse large B-cell lymphoma. Nuclear staining for c-Rel, a pattern consistent with NFkappaB activation, was observed in the Reed-Sternberg cells in 23 of 25 cases of classical Hodgkin lymphoma but only in 1 of 15 cases of anaplastic large cell lymphoma and 3 of 15 cases of nodular lymphocyte predominant Hodgkin lymphoma. A heterogeneous pattern of subcellular c-Rel localization was found in nonmediastinal diffuse large B-cell lymphoma. Taken together, the combination of strong cytoplasmic TRAF1 expression and nuclear c-Rel was present in 80% of cases of classical Hodgkin lymphoma (n = 25) but in only 3% of cases of the other malignant lymphomas tested (n = 62). Thus, the differential expression patterns of downstream components in the CD30 signaling pathway may prove a useful adjunct in distinguishing cases of classical Hodgkin lymphoma from other malignant lymphomas in routine clinical practice.