Background and Aims: One-kilobase sequence of the upstream fragment of the pig lactase-phlorizin hydrolase gene has been shown to control small intestinal-specific expression and postweaning decline of lactase-phlorizin hydrolase in transgenic mice. The aim of this study was to identify the regulatory DNA elements and transcription factors controlling lactase- phlorizin hydrolase expression. Methods: The activity of different lactase- phlorizin hydrolase promoter fragments was investigated by transfection experiments using Caco-2 cells. Electrophoretic mobility shift assays and supershift analyses were used to characterize the interaction between intestinal transcription factors and the identified regulatory elements. Results: Functional analysis revealed three previously undescribed regulatory regions in the lactase-phlorizin hydrolase promoter: a putative enhancer between -894 and -798 binding hepatocyte nuclear factor (HNF)-1 at position - 894 to -880; a repressor-binding element between -278 to -264 to which an HNF-3-like factor is able to bind; and an element between -178 to -164 that binds an activating transcription factor. Conclusions: Identification of three new regulatory regions and HN-1 and HNF-3-like transcription factor as players in the regulation of lactase-phlorizin hydrolase gene transcription has an impact on the understanding of the molecular mechanisms behind age- dependent, tissue-specific, differentiation-dependent, and regional regulation of expression in the intestine.