We recently reported that the apparently non-DNA-binding 65 kd subunit (p65) of the NF-kappa B transcription factor can modulate the DNA-binding specificity of the 50 kd subunit (p50) of NF-kappa B. In this study we provide an explanation for this property of p65. In electrophoretic mobility shift assays and upon UV cross-linking to DNA, gel-purified p65 is shown to be a kappa B-specific DNA-binding protein on its own. The binding activity was only detectable if high amounts of p65 were used for the analyses and after the application of a modified renaturation protocol. DNA-binding of the p65 dimer, in contrast to that of p50, was inhibited by I kappa B-alpha and -beta. This finding is consistent with a receptor function of p65 for both inhibitory subunits. Direct UV cross-linking of NF-kappa B to DNA probes which were photoreactive within only one half-site and a binding competition analysis with p65 showed that p65 has a strong preference for binding to the less conserved half site of kappa B motifs whereas p50 has a moderate preference for the more highly conserved half site. In electrophoretic mobility shift assays and upon sedimentation through glycerol gradients, NF-kappa B appears to exist as a heterodimer composed of one p50 and one p65 subunit whereas data from gel filtration suggest a higher order complex.