I kappa B is an inhibitory protein that stabilizes the inducible cytoplasmic form of the NF-kappa B transcription factor. We have purified I kappa B-alpha, a major form of I kappa B with an apparent molecular size of 37 kd, from cytosol of human placenta. A second chromatographically distinct form, I kappa B-beta, was partially purified and found to be more basic and 3-8 kd larger than the alpha form. The occurrence of distinct forms of I kappa B could explain how NF-kappa B can be activated in response to various agents that signal via different intracellular messenger systems. Both I kappa B-alpha and -beta exclusively inactivated NF-kappa B containing the non-DNA binding 65 kd subunit and, within minutes, could dissociate a high affinity complex of NF-kappa B with its cognate DNA. On the assumption that free I kappa B-alpha and -beta can enter the nucleus, these proteins could rapidly release NF-kappa B from high affinity binding sites in enhancer and promoter elements, thereby terminating NF-kappa B-dependent initiation of gene expression.