We demonstrate, for the first time, that the transcription factor NF-kappaB is constitutively activated during human cervical cancer progression. Immunohistochemical analysis was done using 106 paraffin-embedded cervical tissue specimens of different histological grades. In normal cervical tissue and low-grade squamous intraepithelial lesions, p50, RelA and IkappaB-alpha were mainly localized in the cytosol, whereas in high-grade lesions and squamous cell carcinomas, p50-RelA heterodimers translocated into the nucleus with a concurrent decrease in IkappaB-alpha protein. By Western blot analysis, p50 and RelA were detectable mainly in the cytosolic and nuclear extracts in normal and cancer tissues, respectively, and cytosolic IkappaB-alpha expression was detectable in normal but not in cancer cervical tissues. NF-kappaB DNA-binding activity increased during cervical cancer progression and the binding complex was mainly composed of the p50-RelA heterodimers as revealed by electrophoretic mobility shift assays. Semiquantitative RT-PCR analysis, however, showed increased levels of IkappaB-alpha mRNA in cancer samples presumably because of feedback regulation as a result of enhanced NF-kappaB DNA-binding activity and a consequent functional activation of NF-kappaB. Further immunohistochemical analysis with an antibody to phospho IkappaB-alpha revealed that phosphorylation occurs mainly in squamous intraepithelial lesions, suggesting that the IkappaB-alpha gets phosphorylated initially and degraded as the tumor progressed.