The hepatic nuclear factor 3γ (Hnf3g) is a member of the winged helix gene family of transcription factors and is thought to be involved in anterior–posterior regionalization of the primitive gut. In this study, cis-regulatory elements essential for the expression of Hnf3g in vivo have been characterized. To this end, a 170 kb yeast artificial chromosome (YAC) carrying the entire Hnf3g locus was isolated and modified with a lacZ reporter gene. The two mouse lines carrying the unfragmented Hnf3g–lacZ YAC showed tissue-specific, copy number-dependent and position-independent expression, proving that 170 kb of the Hnf3g locus contain all elements important in the regulation of Hnf3g. Cis-regulatory elements necessary for expression of Hnf3g were identified in a three-step procedure. First, DNase I hypersensitive site mapping was used to delineate important chromatin regions around the gene required for tissue-specific activation of Hnf3g. Second, plasmid-derived transgenes and gene targeting of the endogenous Hnf3g gene locus were used to demonstrate that the 3′-flanking region of the gene is necessary and sufficient to direct reporter gene expression in liver, pancreas, stomach and small intestine. Third, a binding site for HNF-1α and β, factors expressed in organs derived from the endoderm such as liver, gut and pancreas, was identified in this 3′-enhancer and shown to be crucial for enhancer function in vitro. Based on its expression pattern we inferred that HNF-1β is a likely candidate for directly activating Hnf3g gene expression during development.