Isolated and cultured periportal (PP) and pericentral (PC) hepatocytes were used for studying the acinar distribution of several phase I and phase II drug metabolizing reactions and their induction by phenobarbital (PB) and 3-methylcholanthrene (MC) or a combination thereof. Ethoxycoumarin-o-deethylase (EC 1.14.14.1) (ECOD) activity was found to predominate in PC hepatocytes even after induction with MC and PB. Metabolism of biphenyl to a monosulfated product also predominated in PC hepatocytes as did the conversion of harmine to harmine glucuronide and sulfate. In contrast, metabolism of lonazolac was not zonated. The metabolism of all three substrates declined during cultivation for 24 hr and was differentially induced (biphenyl and harmine) or not affected (lonazolac) by MC or PB. Metabolic heterogeneity was best maintained by the combination of MC and PB indicating that the zonal differences are due to a specific balance of phase I and phase II reactions. Glutathione-S-transferase (GST) activities against 1-chloro-2, 4-dinitrobenzene (CDNB) were higher in PC hepatocytes and remained so after induction with PB. In contrast, GST activities against 1,2-dichloro-4-nitrobenzene (DCNB) were almost twice as high in PP cells but equilibrated due to a spontaneous increase during cultivation, particularly in PC hepatocytes. In the presence of PB or both, MC and PB, induction of the activity against DCNB occurred exclusively in the PP hepatocytes. The distribution of the GST subunits Ya and Yb1 roughly corresponded to the pattern of GST activities against CDNB and DCNB, respectively. These results agree with earlier reports demonstrating that PP and PC hepatocytes show different patterns of phase I and phase II drug metabolizing enzymes which are maintained during short-term cultivation. In vitro induction of these activities does not result in equilibration but rather maintenance or even pronounciation of these zonal differences.