We previously showed that mouse alpha-fetoprotein (AFP) enhancer 3 activity is highly restricted to pericentral hepatocytes in the adult liver. Here, using transgenic mice, we show that the upstream enhancer of the rat glutamine synthetase gene is also active, specifically in pericentral regions. Activity of both enhancers is lost in the absence of β-catenin, a key regulator of zonal gene expression in the adult liver. Both enhancers contain a single, highly conserved T-cell factor/lymphoid enhancer factor binding site that is required for responsiveness to β-catenin. We also show that endogenous AFP messenger RNA levels in the perinatal liver are lower when β-catenin is reduced. Conclusion: These data identify the first distinct zonally active regulatory regions required for β-catenin responsiveness in the adult liver, and suggest that postnatal AFP repression and the establishment of zonal regulation are controlled, at least in part, by the same factors. (HEPATOLOGY 2012;56:1892–1901)