To clarify the mechanism underlying regulation of MUC6 expression, we isolated the 5' flanking region of the MUC6 gene (5'-MUC6). We determined the transcription start site of the MUC6 gene, and found a TATA box at -35 to -29bp, a putative NFkappaB consensus sequence at -173 to -164bp, and putative Sp family binding sites at -530 to -521 and -847 to -838bp. The luciferase activity of 5'-MUC6 gradually decreased with deletion of these sites. NFkappaB inhibitory factor IkappaB decreased the luciferase activity, and forced expression of NFkappaB induced MUC6 transcription. An inhibitor of Sp family binding, mithramycin A, suppressed MUC6 transcripts, and Sp1 and Sp3 overexpression up-regulated them. Binding of Sp family members to their putative sites was confirmed by electrophoretic mobility shift assays. Our results suggest that MUC6 transcription is regulated by NFkappaB and Sp family members.