Phenobarbital is a potent inducer of several liver-specific genes such as those encoding detoxication enzymes, including cytochromes P450. However, the mechanisms of action of the barbiturate are poorly understood. Since both, phenobarbital and glucocorticoids, are capable of inducing the same cytochrome P450 species, we asked whether the glucocorticoid receptor could participate to the phenobarbital induced responses. The results presented here show that phenobarbital was able to induce a two-fold increase in the affinity of the glucocorticoid receptor for the binding of dexamethasone, as well as a 30% increase of the receptor number in Reuber rat hepatoma cells of the Fao line. These effects may have a biological significance since they were paralleled by an enhancement of the dexamethasone-induced tyrosine aminotransferase activity, a glucocorticoid inducible function in rat hepatoma cells and in rat liver. To our knowledge, phenobarbital is the first compound shown to be able to induce, in intact cells, an increase in the affinity of the glucocorticoid receptor for the binding of its ligand.