Gut-enriched Krüppel-like factor (GKLF, KLF4) is an epithelial-specific transcription factor whose expression is associated with growth arrest. In order to understand the mechanisms regulating expression of the gene encoding GKLF, we isolated a genomic clone containing murine GKLF. The gene spans 5.3 kb and contains four exons. A major start site of transcription was mapped to an adenine residue 601 nt 5' of the translation initiation codon. An additional 1 kb of the 5'-flanking region was sequenced and found to contain multiple cis -elements homologous to the binding sites of several established transcription factors including Sp1, AP-1, Cdx, GATA, and USF. In particular, three closely spaced GC-boxes 5' of the TATA box resemble the established binding site for GKLF. DNase I protection and electrophoretic mobility shift assays verified that recombinant GKLF bound to each of the three GC-boxes. In co-transfection experiments, GKLF transactivated a reporter gene linked to the GKLF 1 kb 5'-flanking region, as did Sp1, Sp3 and Cdx-2. Mutations of one or both of the first and second GC-boxes in the promoter resulted in diminished transactivation by GKLF. These results demonstrate that the 5'-flanking sequence of the mouse GKLF gene functions as a promoter and is subject to autoregulation by its own gene product.