The transcription factor NF-kappa B is sequestered in the cytoplasm by the inhibitor protein I kappa B alpha. Extracellular inducers of NF-kappa B activate signal transduction pathways that result in the phosphorylation and subsequent degradation of I kappa B alpha. At present, the link between phosphorylation of I kappa B alpha and its degradation is not understood. In this report we provide evidence that phosphorylation of serine residues 32 and 36 of I kappa B alpha targets the protein to the ubiquitin-proteasome pathway. I kappa B alpha is ubiquitinated in vivo and in vitro following phosphorylation, and mutations that abolish phosphorylation and degradation of I kappa B alpha in vivo prevent ubiquitination in vitro. Ubiquitinated I kappa B alpha remains associated with NF-kappa B, and the bound I kappa B alpha is degraded by the 26S proteasome. Thus, ubiquitination provides a mechanistic link between phosphorylation and degradation of I kappa B alpha.