The prodynorphin gene contains several κB motifs, suggesting that κB-specific DNA-binding factors may regulate its expression. Prodynorphin is known to be expressed in human tumor cell lines [Geijer et al., Regul. Peptides, 34 (1991) 181–188] and we report here that several DNA-binding factors of the NF-κB/c-Rel-family are present in the same cells. Three main κB-specific factors, presumably a p50 homodimer, NF-κB which is a p50/p65 heterodimer and a p65/c-Rel heterodimer were identified using an electromobility shift assay (EMSA), immunoabsorption and UV cross-linking experiments. Minor factors consisting of a novel κB-specific protein of about 125 kDa (p125) or being hetero-oligomeric, composed of p125 and either of three other subunits, namely p50, p65 and c-Rel, were also identified. The homo-oligomer of p125 may be identical to the κB-specific factor BETA, previously found only in brain [Korner et al., Neuron, 3 (1989) 563–572]. Comparison of prodynorphin mRNA levels with levels of the κB-specific DNA-binding factors revealed a negative correlation with the level of p50 homodimer, and a positive correlation with the ratio of the levels of p65/c-Rel to NF-κB. No association was found with proenkephalin mRNA levels which were significant in only one cell line. The p50 homodimer, but not p65/c-Rel and NF-κB, bound specifically to a DNA-motif within the dynorphin A-encoding gene sequence. This sequence is located in exon 4 and similar to the consensus κB-sequence. The dynorphin A-encoding sequence may represent an intragenic target for the p50 homodimer, which when bound to the sequence suppresses transcription.