The cytochrome P450 gene CYP2H1 is highly induced by phenobarbital in chick embryo hepatocytes. Recent studies have established that the orphan nuclear receptor CAR plays a critical role in the induction mechanism. Here, we show that a high concentration of the potent glucocorticoid and progesterone receptor antagonist RU486 almost completely blocks phenobarbital-induced accumulation of CYP2H1 mRNA in hepatocytes yet has no effect on basal expression. In marked contrast, CYP2H1 mRNA induced by the phenobarbital-type inducers glutethimide and 2-allylisopropylacetamide is not affected by RU486. RU486 inhibition is not mediated through the glucocorticoid or progesterone receptors. Transient transfection studies showed that RU486 does not repress through activation of the orphan receptor PXR and subsequent competition with CAR for binding to the upstream drug-responsive 556-base-pair enhancer. Additionally, none of the known functional transcription factor binding sites found in the enhancer region was a target of RU486 inhibition. Using an artificial construct containing multiple CAR binding sites, we also established that RU486 has no direct effect on the activity of exogenously expressed CAR. There is no evidence that phenobarbital binds to CAR; we propose that RU486 inhibits phenobarbital induction, either by interfering with a phenobarbital-dependent mechanism responsible for nuclear import of CAR or with the metabolism of phenobarbital to the true inducer. Whether a novel nuclear receptor that binds RU486 at high concentrations plays a role in the inhibitory action of RU486 is an interesting possibility.