The neuropoietic cytokine ciliary neurotrophic factor (CNTF) potently induces transcription of the vasoactive intestinal peptide (VIP) gene through a 180-base pair (bp) cytokine response element (CyRE) in the VIP promoter. We have previously shown that CNTF induction of STAT and AP-1 protein binding within the CyRE is necessary to mediate CNTF induction of VIP gene transcription. We now show that a third, previously uncharacterized site at the 3'-end of the CyRE is also critical to CNTF induction of CyRE transcription. A 4-bp mutation in this 3'-region reduced CNTF-mediated induction of transcription approximately 80%. Whereas mutations in both the STAT and AP-1 sites substantially reduced CNTF induction of transcription, mutations in these sites together with the novel 3'-site completely abolished the ability of CNTF to induce CyRE-mediated transcription. Gel shift analysis indicated that a complex in neuroblastoma cells bound specifically to this 3'-site. This complex was not altered by CNTF treatment. Mutations in an 8-bp sequence (TTACTGGA) eliminated binding of this protein complex and markedly reduced transcriptional activation of the CyRE by CNTF. Thus, we have identified a protein complex binding to a novel DNA sequence that is necessary for full CNTF induction of VIP gene transcription.