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The cellular microenvironment plays an important role in liver zonation, the spatial distribution of metabolic tasks amongst hepatocytes lining the sinusoid. Standard tissue culture practices provide an excess of oxygen and a lack of signaling molecules typically found in the liver. We hypothesized that incorporating physiologically relevant environments would promote post-differentiation patterning of hepatocytes and result in zonal-like characteristics. To test this hypothesis, we evaluated the transcriptional regulation and activity of drug-metabolizing enzymes in HepaRG cells exposed to three different oxygen tensions, in the presence or absence of Wnt/β-catenin signaling. The drug-metabolizing activity of cells exposed to representative periportal (11% O) or perivenous (5% O) oxygen tensions were significantly less than cells exposed to ambient oxygen. A comparison of cytochrome P450 (CYP) 1A2, 2D6, and 3A4 activity at PP and PV oxygen tensions showed significant increases at the lower oxygen tension. The activation of the Wnt/β-catenin pathway only modestly impacted CYP activity at PV oxygen tension, despite a significant increase in expression under this condition. Our results suggest oxygen tension is the major contributor to zonal patterning in HepaRG cells, with the Wnt/β-catenin signaling pathway playing a lesser albeit important role. Our datasets also highlight the importance of including activity-based assays, as transcript data alone does not provide an accurate picture of metabolic competence. This work investigates the post-differentiation patterning of HepaRG cells cultured at physiologically relevant oxygen tensions, in the presence and absence of Wnt/β-catenin signaling. HepaRG cells exposed to periportal (11% O) or perivenous (5% O) oxygen tensions display zonation-like patterning of both cytochrome P450 (CYP) and glucuronosyltransferase (UGT) enzymes. These datasets also suggest that oxygen is a primary regulator of post-differentiation patterning, with Wnt/β-catenin having a lesser effect on activity but a significant effect on transcriptional regulation of these enzymes.