Biochemical studies have shown that microsomes represent an important subcellular fraction for determining 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) effects. Proteomic analysis by two-dimensional gel-mass spectrometry of liver microsomes was undertaken to gain new insight into the actions of TCDD in male and female rats. Proteomic analysis showed TCDD induced several xenobiotic metabolism enzymes as well as a protein at 90 kDa identified by mass spectrometry as IκB kinase β/IKK2. This observation led to the discovery of other NF-κB binding proteins and kinases in microsomes and effects by TCDD. Western blotting for IKK and IκB family members in microsomes showed a distinct pattern from cytosol. IKK1 and IKK2 were both present in microsomes and were catalytically active although, unlike cytosol, IKKγ/NEMO was not detectable. TCDD exposure produced an elevation in cytosolic and microsomal IKK activity of both genders. The NF-κB binding proteins IκBβ and IκBγ were prevalent in microsomes, while IκBα and IκBε proteins were absent. TCDD treatment produced hyperphosphorylation of microsomal IκBβ in both sexes with females being most sensitive. In cytosol, IκBα, IκBβ, and IκBε, but not IκBγ, were clearly observed but were not changed by TCDD. Overall, proteomic analysis indicated the presence of NF-κB pathway members in microsomes, selectively altered by dioxin, which may influence immune and inflammatory responses within the liver.