In mammalian liver, high glutamine synthetase (GS) expression is restricted to hepatocytes surrounding the terminal venules. The most important enhancer of the GS gene is located approximately 2520 base pairs (bp) upstream from the transcriptional start point. The nature of the transcription factors that bind to the enhancers has remained enigmatic. In this study, we purified nuclear proteins binding to the element. Supershift assays and footprint experiments with purified protein identified activated STAT5 as a transcription factor binding to a site within the enhancer. In addition, a second binding site close to the STAT5 site was observed that also binds a protein present in nuclear extracts. Sequence analysis indicated that the second site may bind a member of the LEF/TCF transcription factor family. Reporter gene assays demonstrate that the STAT5 binding site mediates enhancement of expression whereas the LEF/TCF site functions as a silencer of growth hormone-mediated enhancement in normal hepatocytes. LEF/TCF-sites are known to function as silencers in the absence and as enhancers in the presence of activated beta-catenin. In conclusion, the GS 5' enhancer contains elements important for GS expression in cells carrying an activated form of beta-catenin as previously shown in experimentally induced hepatocellular carcinomas.