The immunoglobulin kappa light chain enhancer, kappa B, is an important cis-acting transcriptional element. kappa B binds a number of proteins including the members of the ubiquitous NF-kappa B family of transcription factors. Agarose beads coupled to a double-stranded oligonucleotide containing the kappa B motif were used to isolate a 65-kDa predominantly nuclear phosphoprotein. Southwestern blot analysis demonstrated that this phosphoprotein can bind the kappa B element directly and specifically. This kappa B-associated protein was phosphorylated in vivo and in vitro by a nuclear serine/threonine kinase(s) which, in a number of different cell lines, appeared to be stimulated in response to interleukin-1 alpha and lipopolysaccharide treatment. In the B cell lines 70Z/3 and CH12 LX2B, and the T cell line EL-4 6.1 C10 the activity of the kappa B-associated kinase(s) correlated with the binding activity of nuclear NF-kappa B displayed in a gel shift assay. In vitro, the 65-kDa protein was phosphorylated in the absence of exogenously added kinase. The 65-kDa phosphoprotein and the kinase activity remained associated following sequential anion-exchange and hydrophobic interaction chromatography. These results suggest that the kappa B-associated phosphoprotein is either autophosphorylated or is phosphorylated by a closely associated kinase(s). Stimulation of a nuclear protein kinase which is closely associated with a sequence-specific DNA may reflect a novel mechanism by which growth factors regulate gene expression.