Comparative genomic hybridization (CGH) analysis of DNA extracted from a diffuse lymphoma with a large cell component (DLLC) that displayed double minute chromosomes upon conventional karyotypic analysis indicated overt amplification of DNA sequences derived from the 2p13–15 region. Southern blot analysis of this tumor DNA with a cDNA probe for the proto-oncogene REL, previously mapped to 2p14–15, indicated a greater than 35-fold amplification of REL. To determine the incidence of REL amplification and possible clinical or histologic association with DLLC, a panel of 111 tumor DNAs from DLLC specimens was screened for REL amplification by Southern blot analysis. A copy number of > or = 4 was noted in 26 cases (23%). Southern blot analysis of these 26 tumor DNAs with a cDNA probe for TGFA, mapped to 2p13, indicated lack of coamplification except in one case. Another member of the Rel/NF- kappa B family of transcriptional activators, RELA/p65 mapped to 11q13, was amplified in five cases as determined by Southern blot analysis using a cDNA probe. Nineteen of the 26 DLLC (73%) with REL amplification were primary extranodal lymphomas. As a group, the tumors with REL amplification demonstrated an increased frequency of chromosomal aberrations previously associated with tumor progression, suggesting an oncogenic effect of amplified REL in B-lymphoid cells that already contained a transforming genetic lesion. Thus, REL amplification is a frequent event in DLLC, and probably constitutes a progression-associated marker of primary extranodal lymphomas. This study shows the usefulness of the CGH technique in identifying chromosomal regions overrepresented in tumors that can point to amplified genes and may be correlated with clinical features of the disease.