We determined the extent to which diurnal variation in cholesterol synthesis in liver is controlled by steady-state mRNA levels for the rate-limiting enzyme in the pathway, hydroxymethylglutaryl (HMG)-CoA reductase. Rats 30 days of age and maintained on a low-cholesterol diet since weaning were injected intraperitoneally with (3)H(2)O. The specific radioactivity of the whole-body water pool soon became constant, allowing for expression of values for incorporation of label into cholesterol as absolute rates of cholesterol synthesis. In liver, there was a peak of cholesterol synthesis from 8 pm to midnight, a 4-fold increase over synthesis rates from 8 am to noon. Increases in synthesis were quantitatively in lock step with increases in mRNA levels for HMG-CoA reductase occurring 4 h earlier. In a parallel experiment, rats received 1% cholesterol in the diet from weaning to 30 days of age. Basal levels of hepatic cholesterol synthesis were greatly diminished and there was little diurnal variation of cholesterol synthesis or of levels of mRNA for HMG-CoA reductase. Levels of mRNA for the low density lipoprotein receptor and scavenger receptor-B1 (putative high density lipoprotein receptor) showed little diurnal variation, regardless of diet. This suggests that diurnal variation of hepatic cholesterol synthesis is driven primarily by varying the steady-state mRNA levels for HMG-CoA reductase. Other tissues were also examined. Adrenal gland also showed a 4-fold diurnal increase in accumulation of recently synthesized cholesterol. In contrast to liver, however, there was little corresponding change in mRNA expression for HMG-CoA reductase. Much of this newly synthesized cholesterol may be of hepatic origin, imported into adrenal by SR-B1, whose mRNA was up-regulated 2-fold. In brain, there was no diurnal variation in either cholesterol synthesis or mRNA expression, and no influence of high- or low-cholesterol diets on synthesis rates or HMG-CoA reductase mRNA levels.