Selective inhibition of proinflammatory chemokines such as IL-8 is an important approach to combat inflammatory and infection diseases. Previous studies suggested that interaction of transcription factors NFkappaB repressing factor (NRF) and NFkappaB play a crucial role in activation of IL-8 gene expression. In a search for a specific inhibitor of IL-8 expression, we applied tandem affinity purification to investigate interaction of NRF and NFkappaB p65 in cells. We identified a synthetic peptide corresponding to aa 223-238 of NRF interfering with binding of endogenous p65 to NRF. Furthermore, nucleofection experiments were established to introduce this inhibitory peptide into the nucleus of IL-1 stimulated human cervical and Helicobacter pylori infected gastric epithelial cells. Our data clearly show that the specific peptide disturbing NRF/NFkappaB interaction is able to significantly decrease endogenous IL-8 gene transcription in response to IL-1 or Helicobacter pylori infection. Thus, our study provides novel insights into NRF and NFkappaB interaction in vivo and may facilitate the design of new anti-IL-8 drugs based on novel strategies.