Hepatocyte nuclear factor-1 (HNF-1) is a modified homeodomain-containing transcription factor that has been implicated in the regulation of intestinal genes. To define the importance and underlying mechanism of HNF-1 for the regulation of intestinal gene expression in vivo, we analyzed the expression of the intestinal differentiation markers and putative HNF-1 targets, lactase-phlorizin hydrolase (LPH) and sucrase-isomaltase (SI), in hnf1 null mice. We found that in adult jejunum, LPH mRNA in hnf1-/- mice was reduced 95% as compared to wild-type controls (P<0.01, n=4), whereas SI mRNA was virtually identical to that in wild-type mice. Further, SI mRNA abundance was unchanged in the absence of HNF-1 along the length of the adult mouse small intestine as well as in newborn jejunum. We found that HNF-1 occupies the promoters of both the LPH and SI genes in vivo. However, in contrast to liver and pancreas where HNF-1 regulates target genes by recruitment of histone acetyl transferase activity to the promoter, the histone acetylation state of the LPH and SI promoters was not affected by the presence or absence of HNF-1. Finally, we show that a subset of hypothesized intestinal target genes are regulated by HNF-1 in vivo, and this regulation occurs in a defined tissue-specific and developmental context. These data indicate that HNF-1 is an activator of a subset of intestinal genes, and induces these genes through an alternative mechanism in which it is dispensable for chromatin remodeling.