Chronic treatment of rats with phenobarbital has been reported to decrease gluconeogenesis in rat hepatocytes by a 50% inhibition of phosphoenolpyruvate (P-pyruvate) carboxykinase activity [Argaud, D., Halimi, S., Catelloni, F. & Leverve, X. (1991) Biochem. J. 280, 663-669]. Contrary to the current knowledge of P-pyruvate carboxykinase regulation, we failed to find a diminution of either P-pyruvate carboxykinase protein (by using a polyclonal antibody) or P-pyruvate carboxykinase mRNA, in the liver of rats treated with phenobarbital for 2 weeks. Kinetic studies of P-pyruvate carboxykinase activity, measured by either carboxylation of P-pyruvate or decarboxylation of oxaloacetate, revealed a decrease in both V(max) and Km after phenobarbital treatment, whereas the nutritional state affected only the V(max), as expected. Assessment of P-pyruvate carboxykinase specificity was confirmed by the full inhibition of the enzyme with its specific inhibitor 3-mercaptopicolinate in the micromolar range. P-Pyruvate carboxykinase, purified either by ammonium sulfate fractionation or by immunoprecipitation, exhibited a similar decrease in affinity after phenobarbital treatment. Although the molecular mass does not appear to be altered, the pH sensitivity to 3-mercaptopicolinate inhibition and the enzyme recovery after immunoprecipitation both seemed to be affected. This leads us to propose that the effect of chronic phenobarbital treatment on P-pyruvate carboxykinase activity is not the result of transcriptional regulation but is exerted at the post-translational level.