We have examined the influence of the phenobarbital-induced proliferation of the hepatic endoplasmic reticulum (ER) on the activities of the components of the glucose-6-phosphatase system, i.e., the enzyme, the glucose-6-P translocase (T1), and the phosphate translocase (T2). Young male rats were injected ip twice daily for 4 days with 4 mg/100 g body wt of phenobarbital (PB) or an equivalent volume of saline solution. On the fifth day, the rats were killed and smooth (SER) and rough (RER) fractions of the ER were isolated from liver homogenates. Kinetic constants for glucose-6-P hydrolysis by the system and enzyme were determined and used to calculate the kinetic constants for glucose-6-P transport. T2 activity was approximated by assaying the pyrophosphatase activity at pH 6.0 in intact microsomes. Three times more SER protein was recovered from livers of PB-treated rats. PB-treatment did not alter total liver enzyme activity, but total liver T1 activity was decreased to 59% of the control value. Maximal specific activities of the system, enzyme and T1 were all reduced by PB treatment to 44% of control values in the RER and to 68% of control values in the SER. PB treatment reduced the apparent activity of T2 in RER and SER to 35 and 49% of the respective control values. In the SER from both groups of rats, T1 activity or apparent T2 activity divided by enzyme activity was about 55% of the corresponding ratio in the RER. Our analysis of these data suggests that the lower activities of T1 and T2 in the smooth ER are the results of suppression by some intrinsic component localized in the smooth membrane. Accordingly, the reduction in total liver T1 activity and, therefore, system activity in PB-treated rats reflects the redistribution of the glucose-6-P translocase from the RER to the more abundant SER membrane where it is less active. The possibility is discussed that a higher cholesterol content within the SER membrane is responsible for the lower transport activities.