The localization of the form of cytochrome P-450 that is induced by phenobarbital was studied by direct immunofluorescence in the hepatocytes of rats pretreated with phenobarbital in comparison with saline-treated rats. Specific fluorescence was seen in the hepatocyte cytoplasm in saline- and phenobarbital-treated rats; a more concentrated halo of fluorescence was detected surrounding the nuclei in the centrilobular zones after phenobarbital treatment. In the saline-treated rats, fluorescence was barely discernible but slightly more intense in the centrilobular than perilobular zones. In phenobarbital-treated rats, fluorescence was much more intense, with a similar but much greater difference between the centrilobular and perilobular zones. The tissue distribution and induction site of this component of the cytochrome P-450-dependent microsomal system may be relevant to the site of drug toxicity and the development of chemical carcinogenesis.