In uninduced cells, the NF-kappa B transcription factor resides in the cytoplasm in complex with an inhibitory protein, I kappa B. I kappa B is a specific inhibitor of DNA binding and apparently prevents nuclear uptake of NF-kappa B. Stimulation of cells, for instance with the cytokine tumor necrosis factor, releases I kappa B and allows nuclear translocation and DNA binding of NF-kappa B to regulatory DNA sequences in many genes. We recently reported on the purification of a major form of I kappa B, referred to as I kappa B-alpha, with a molecular size of 37 kDa. Here, we purified and characterized I kappa B-beta, a chromatographically distinct second form of I kappa B. I kappa B-beta has a size of 43 kDa and, as I kappa B-alpha, an acidic isoelectric point between 4.8 and 5.0. Both forms of I kappa B were inactivated by a treatment with protein kinases A and C in vitro. In contrast to I kappa B-alpha, I kappa B-beta lost its inhibiting activity upon a treatment with phosphatase. Phosphatase treatment also released active NF-kappa B from its inactive complex with I kappa B-beta suggesting that the activation of NF-kappa B in intact cells might not only rely on phosphate transfer onto I kappa B but also on phosphate removal from one form of I kappa B.