NF-kappa B is an inducible transcription factor that regulates the expression of numerous genes involved in immune and inflammation responses and in cellular growth control. Typically, NF-kappa B is localized in the cytoplasm complexed with members of the I kappa B family. The most well characterized form of NF-kappa B is comprised of a heterodimer of a 50 kD (p50/NFKB1) and a 65 kD (p65/RelA) protein. This heterodimeric protein was thought to be primarily responsible for transcriptional regulation of target genes. However, recent studies have led to the identification of other kappa B binding proteins such as c-Rel, RelB and p52 (NFKB2/lyt-10) although their role in gene regulation has been less clear. Here, using gel mobility shift assays as well as a highly sensitive DNA-protein crosslinking assay, we provide evidence for the existence of multiple tumor necrosis factor (TNF)- inducible kappa B binding complexes containing various members of the NF-kappa B/Rel family, namely p50 and p65 as well as the c-Rel and p52 oncoproteins. Dimeric complexes containing various combinations of these proteins appear rapidly in nuclei of TNF-alpha-stimulated cells and include, along with a p50-p65 heterodimer, p50-c-Rel, p65-c-Rel, p52-c-Rel and p52-p65 complexes. The presence of multiple inducible complexes containing distinct combinations of NF-kappa B/Rel family members indicate that specific kappa B responsive genes may be regulated in an NF-kappa B subunit-dependent manner.