To study the molecular mechanisms controlling guanylin expression, we have cloned the mouse guanylin gene, including 2.7 kb of upstream sequence. We show that the first 133 base pairs (bp) of the upstream guanylin promoter are sufficient to drive near maximal (6-fold over basal) luciferase reporter gene expression in Caco-2 intestinal cells; at least 300 bp of upstream promoter are required for reporter gene expression in HT-29 intestinal cell lines. Using electromobility shift assays, we demonstrate that nuclear proteins bind to the hepatocyte nuclear factor-1 (HNF-1) consensus sequence in the guanylin promoter. The HNF-1 consensus sequence, located in the immediate 5' flanking region, is required for transcriptional activation of the guanylin gene in both intestinal cell lines. Mutagenesis of the HNF-1 consensus sequence abolishes transcriptional activation of guanylin promoter-luciferase reporter gene constructs. Cotransfection of these constructs with HNF-1alpha augments transcriptional initiation of the reporter gene. In contrast, HNF-1beta has no significant effect on transcription of the reporter gene. These experiments demonstrate that HNF-1alpha is an important regulatory element in the transcriptional activation of guanylin.