The transcription factor NF-kappa B is maintained in an inactive cytoplasmic state by I kappa B inhibitors. In mammalian cells, I kappa B alpha and I kappa B beta proteins have been purified and shown to be the inhibitors of NF-kappa B through their association with the p65 or c-Rel subunits. In addition, we have isolated a third NF-kappa B inhibitor, I kappa B epsilon (1). Upon treatment with a large variety of inducers, I kappa B alpha, I kappa B beta are proteolytically degraded, resulting in NF-kappa B translocation into the nucleus. Here we show that in E29.1 T cell hybridoma I kappa B alpha and I kappa B beta are equally associated with p65 and that I kappa B beta is degraded in response to TNF alpha in contrast to what has been originally published. Our data also suggest that, unlike I kappa B alpha, I kappa B beta is constitutively phosphorylated and resynthesized as a hypophosphorylated form. The absence of slow migrating forms of I kappa B beta following stimulation suggests that the phosphorylation does not necessarily constitute the signal-induced event which targets the molecule for proteolysis.