Peroxisomes in hepatocytes from mice administered 35, 50, or 100 mg/kg of phenobarbital (PB) were analyzed by quantitative electron microscopy. In perivenular hepatocytes, the volume of peroxisomes per unit cytoplasmic volume decreased to 78 or 57% of that of control animals by administration of 50 or 100 mg/kg. Their average volume also decreased to 78 or 64% of that in control animals in animals injected with 35 or 50 mg/kg. Further, cytochemical catalase activity appeared decreased in peroxisomes of hepatocytes of this zone after administration of 50 or 100 mg/kg. These suggest an inhibitory action of PB administration of biogenesis of peroxisomes in perivenular hepatocytes. In periportal hepatocytes, however, the volume density, average volume, and cytochemical catalase activity of peroxisomes did not change by administration of any doses of PB. Thus, PB administration appears to produce no inhibitory effect on the peroxisome biogenesis in periportal hepatocytes. On the other hand, the number of peroxisomes per unit cytoplasmic volume increased in both periportal and perivenular hepatocytes in animals injected with 50 mg/kg, although it returned to the level of control animals by injection of 100 mg/kg. The inhibitory effect of PB on the peroxisome neogenesis in perivenular hepatocytes may be related to the marked smooth endoplasmic reticulum proliferation by administration of this drug.