In contrast to multiple injections of phenobarbital, a single injection of 50 mg/kg body wt of phenobarbital into immature male rats results, after a transient increase in hepatocyte mitotic activity, in a marked decrease in hepatocyte mitotic activity to below control levels by day 3, followed by a return to control levels by day 5. This unusual pattern of hepatocyte mitotic activity can be called fourth again by a second injection of 50 mg phenobarbital/kg body wt. However, a single injection of 50 mg/kg body wt of phenobarbital into immature male rats results in a pattern of change of liver wet weight, protein, and aminopyrine demethylase activity which is similar to that observed after multiple injections of phenobarbital, except that the changes are smaller in magnitude. Liver wet weight, protein, and aminopyrine demethylase activity increase and reach a peak within two days after phenobarbital injection, and then they return to control levels by five days. The same pattern of change in liver wet weight, protein, and aminopyrine demethylase activity can be elicited again by a second injection of 50 mg phenobarbital/kg body wt.