The human large B-cell lymphoma cell line RC-K8 has a rearranged REL locus that directs the production of a chimeric protein, termed REL-NRG (Non-Rel Gene). In this study, we show that RC-K8 cells have constitutively nuclear heterodimeric and homodimeric DNA-binding complexes that consist of p50, REL, and REL-NRG. In vitro, IkappaBalpha can block the DNA-binding activity of wild-type REL homodimers but not REL-NRG homodimers. In vivo, REL-NRG cannot activate transcription of a kappaB site reporter plasmid, suggesting that it is a transcription repressing or blocking REL protein. By Western blotting, no IkappaBalpha protein can be detected in extracts of RC-K8 cells. The absence of IkappaBalpha protein in RC-K8 cells appears to be due to mutations that cause premature termination of translation in three of the four copies of the IKBA gene in RC-K8 cells. Re-expression of wild-type IkappaBalpha or a super-repressor form of IkappaBalpha in RC-K8 cells is cytotoxic; in contrast, expression of a dominant-negative form of IkappaB kinase does not affect the growth of RC-K8 cells. By cDNA microarray analysis, a number of previously identified Rel/NF-kappaB target genes are overexpressed in RC-K8 cells, consistent with there being transcriptionally active REL complexes. Taken together, our results suggest that the growth of RC-K8 cells is dependent on the activity of nuclear wild-type REL dimers, while the contribution of REL-NRG to the transformed state of RC-K8 cells is less clear. Nevertheless, the RC-K8 cell line is the first tumor cell line identified with mutations in genes encoding multiple proteins in the Rel/NF-kappaB signal transduction pathway.