BCL-6 is a transcription repressor frequently deregulated in non-Hodgkin's B cell lymphomas. Its activity is also critical to germinal center development and balanced Th1/Th2 differentiation. Previous studies have suggested that NF-kappaB activity is suppressed in germinal center and lymphoma B cells that express high levels of BCL-6, and yet the reason for this is unknown. We report in this study that BCL-6 can bind to three sequence motifs in the 5' regulatory region of NF-kappaB1 in vitro and in vivo, and repress NF-kappaB1 transcription both in reporter assays and in lymphoma B cell lines. BCL-6(-/-) mice further confirm the biological relevance of BCL-6-dependent regulation of NF-kappaB1 because BCL-6 inactivation caused notable increase in p105/p50 proteins in several cell types. Among these, BCL-6(-/-) macrophage cell lines displayed a hyperproliferation phenotype that can be reversed by NF-kappaB inhibitors, e.g., N-tosyl-l-phenylalanine chloromethyl ketone and SN50, a result that is consistent with increased nuclear kappaB-binding activity of p50 homodimer and p50/p65 heterodimer. Our results demonstrate that BCL-6 can negatively regulate NF-kappaB1 expression, thereby inhibiting NF-kappaB-mediated cellular functions.