To further specify the cellular origin and nature of anaplastic large-cell lymphoma (ALCL) and its relationship to other lymphoid neoplasms, particularly Hodgkin's disease (HD), we investigated the presence of cytotoxic molecules in a large well-characterized series of these tumors. For expression of the cytotoxic molecules perforin and granzyme B, in situ hybridization (ISH) and immunohistology were used, respectively. Overall, 23 of 25 ALCLs of T/null phenotype and five (three mixed cellularity and two nodular sclerosis) of 57 HD cases showed the presence of perforin transcripts and/or granzyme B molecules in neoplastic cells. Polymerase chain reaction (PCR) analysis of ALCLs showed that most (10 of 11) cases of null-cell ALCL (null-ALCL) contained a clonal rearrangement of T-cell receptor beta-chain genes, as did T-cell ALCL (T-ALCL; 9 of 10 cases). However, both cytotoxic molecules and clonally rearranged T-cell receptor beta-chain genes were absent in seven of seven and eight of nine cases of B-cell ALCL (B-ALCL), respectively. These data show that all or nearly all T-ALCLs, irrespective of the clinical subform or the lack of T-cell-associated molecules, are derived from activated cytotoxic T cells. The same appears to be true for the neoplastic cells of rare HD cases. These findings indicate that T-ALCLs are different from B-ALCLs and the majority of HD cases, and suggest that some HD cases, especially those with T-cell antigen-positive tumor cells, may be closely related to T-ALCL, at least in terms of cellular origin.