In liver, most genes are expressed with a porto-central gradient. The transcription factor hepatic nuclear-factor4α (HNF4α) is associated with 12% of the genes in adult liver, but its involvement in zonation of gene expression has not been investigated. A putative HNF4α-response element in the upstream enhancer of glutamine synthetase (GS), an exclusively pericentral enzyme, was protected against DNase-I and interacted with a protein that is recognized by HNF4α-specific antiserum. Chromatin-immunoprecipitation assays of HNF4α-deficient (H4LivKO) and control (H4Flox) livers with HNF4α antiserum precipitated the GS upstream enhancer DNA only from H4Flox liver. Identical results were obtained with a histone-deacetylase1 (HDAC1) antibody, but antibodies against HDAC3, SMRT and SHP did not precipitate the GS upstream enhancer. In H4Flox liver, GS, ornithine aminotransferase (OAT) and thyroid hormone-receptor β1 (TRβ1) were exclusively expressed in pericentral hepatocytes. In H4LivKO liver, this pericentral expression remained unaffected, but the genes were additionally expressed in the periportal hepatocytes, albeit at a lower level. The expression of the periportal enzyme phosphoenolpyruvate carboxykinase had declined in HNF4α-deficient hepatocytes. GS-negative cells, which were present as single, large hepatocytes or as groups of small cells near portal veins, did express HNF4α. Clusters of very small GS- and HNF4α-negative, and PCNA- and OV6-positive cells near portal veins were contiguous with streaks of brightly HNF4α-positive, OV6-, PCNA-, and PEPCK-dim cells. Conclusion: Our findings show that HNF4α suppresses the expression of pericentral proteins in periportal hepatocytes, possibly via a HDAC1-mediated mechanism. Furthermore, we show that HNF4α deficiency induces foci of regenerating hepatocytes. (HEPATOLOGY 2007;45:433–444.)