Members of the nuclear factor (NF)-κB family of transcription factors play a crucial role in cellular activation, immune responses, and oncogenesis. In most cells, they are kept inactive in the cytosol by complex formation with members of the inhibitor of NF-κB (IκB) family, whose degradation activates NF-κB in response to diverse stimuli. In Hodgkin's lymphoma (HL), high constitutive nuclear activity of NF-κB is characteristic of the malignant Hodgkin and Reed-Sternberg (H/RS) cells, which occur at low number in a background of nonneoplastic inflammatory cells. In single H/RS cells micromanipulated from histological sections of HL, we detect clonal deleterious somatic mutations in the IκBα gene in two of three Epstein-Barr virus (EBV)-negative cases but not in two EBV-positive cases (in which a viral oncogene may account for NF-κB activation). There was no evidence for IκBα mutations in two non-HL entities or in normal germinal center B cells. This study establishes deleterious IκBα mutations as the first recurrent genetic defect found in H/RS cells, indicating a role of IκBα defects in the pathogenesis of HL and implying that IκBα is a tumor suppressor gene.