The pregnane X receptor (PXR) was isolated as a xenobiotic receptor that regulates responses to various xenobiotic agents. In this study, we show that PXR plays an important endobiotic role in adrenal steroid homeostasis. Activation of PXR by genetic (transgene) or pharmacological (ligand, such as rifampicin) markedly increased plasma concentrations of corticosterone and aldosterone, the respective primary glucocorticoid and mineralocorticoid in rodents. The increased levels of corticosterone and aldosterone were associated with activation of adrenal steroidogenic enzymes, including cytochrome P450 (CYP)11a1, CYP11b1, CYP11b2, and 3beta-hydroxysteroid dehydrogenase. The PXR-activating transgenic mice also exhibited hypertrophy of the adrenal cortex, loss of glucocorticoid circadian rhythm, and lack of glucocorticoid responses to psychogenic stress. Interestingly, the transgenic mice had normal pituitary secretion of ACTH and the corticosterone-suppressing effect of dexamethasone was intact, suggesting a functional hypothalamus-pituitary-adrenal axis despite a severe disruption of adrenal steroid homeostasis. The ACTH-independent hypercortisolism in the PXR-activating transgenic mice is reminiscent of the pseudo-Cushing's syndrome in patients. The glucocorticoid effect appears to be PXR specific, as the activation of constitutive androstane receptor in transgenic mice had little effect. We propose that PXR is a potential endocrine disrupting factor that may have broad implications in steroid homeostasis and drug-hormone interactions.