A recent study has shown that the dimeric form of the 50-kD DNA-binding subunit of the NF-kappa B transcription factor is commonly associated with two molecules of a 65-kD protein (p65) and that p65 appears to be required for inactivation of the p50 dimer by the inhibitory subunit I kappa B. Here, we provide evidence that p65 serves as a receptor for I kappa B. Preincubation of I kappa B with purified p65 prevented I kappa B from inactivating the heterotetrameric form of NF-kappa B. Furthermore, excess p65 could very efficiently activate the latent form of NF-kappa B composed of p50, p65, and I kappa B, presumably by binding the I kappa B, which was released from the complex due to its inherent off rate. An additional function of p65 in modulating the DNA-binding specificity of p50 was found. In the absence of p65, p50 could bind with high affinity to completely palindromic sites. The heterotetramer recognized these sites with an affinity greater than 10-fold lower but preferred the less symmetric physiological kappa B site. The affinity of p50 for the most frequent kappa B motif 5'-GGGACTTTCC-3' was enhanced twofold by p65 to yield a dissociation constant of approximately 4 x 10(-13) M. This study describes novel functions for a non-DNA-binding accessory protein of a transcription factor in controlling its inducibility and DNA-binding specificity.