Cytochrome P450 3A4 (CYP3A4) is responsible for oxidative metabolism of more than 60% of all pharmaceuticals. CYP3A4 is inducible by xenobiotics that activate pregnane X receptor (PXR), and enhanced CYP3A4 activity has been implicated in adverse drug interactions. Recent evidence suggest that the widely used plasticizer, di-2-ethylhexyl phthalate (DEHP), and its primary metabolite mono-2-ethylhexyl phthalate (MEHP) may act as agonists for PXR. Hospital patients are uniquely exposed to high levels of DEHP as well as being administered glucocorticoids. Glucocorticoids positively regulate PXR expression in a glucocorticoid receptor (GR)-mediated mechanism. We suggest that the magnitude of CYP3A4 induction by phthalates is dependent on the expression of PXR and may be significantly higher in the presence of glucocorticoids. DEHP and MEHP induced PXR-mediated transcription of the CYP3A4 promoter in a dose-dependent fashion. Coexposure to phthalates and dexamethasone (Dex) resulted in enhanced CYP3A4 promoter activity; furthermore, this induction was abrogated by both the GR antagonist RU486 and GR small interfering ribonucleic acid. Dex induced PXR protein expression in human hepatocytes and a liver-derived rat cell line. CYP3A4 protein was highly induced by Dex and DEHP coadministration in human hepatocyte cultures. Finally, enhanced 6beta-hydroxytestosterone formation in Dex and phthalate cotreated human hepatocytes confirmed CYP3A4 enzyme induction. Concomitant exposure to glucocorticoids and phthalates resulting in enhanced metabolic activity of CYP3A4 may play a role in altered efficacy of pharmaceutical agents. Understanding the role of glucocorticoid regulation of PXR as a key determinant in the magnitude of CYP3A4 induction by xenobiotics may provide insight into adverse drug effects in a sensitive population.