The objective of this study was to determine whether the inductive effect of phenobarbital (PB) on liver cytochrome P-450 was the result of the action of this drug on all or some hepatocytes. For this purpose, a light (cell band I) and a heavy (cell band II) subpopulation of hepatocytes were separated from rat liver in a continuous density gradient. To determine the location of these hepatocytes in tissue, [14C]bromobenzene, which binds covalently to centrilobular hepatocytes, was administered. The specific activity (14C dpm/mg protein) was greater in cells of band I than in cells of band II, suggesting a predominant contribution of centrilobular hepatocytes to the lighter cell band. Microsomes were separated from each cell subpopulation after 3 days of PB administration and cytochrome P-450 was measured. Although a fivefold increment in cytochrome P-450 content of light hepatocytes was noted, the content of heavy hepatocytes was similar to that of the respective subpopulation in controls. Concomitantly, PB administered for 3 days induced the smooth endoplasmic reticulum of centrilobular hepatocytes only, as revealed by electron microscopy of whole tissue. These results indicated that PB induces cytochrome P-450 in a selective subpopulation of hepatocytes, most likely located near the terminal hepatic venule.