We studied the cholesterol and phospholipid content and the cholesterol-to-phospholipid molar ratio of the small intestinal microvillus membrane in fetal, weanling, and adult rats. We also investigated the effect of glucocorticoid administration on these lipids and the ontogeny of 1,2-diglycerol-CDP choline phosphocholine transferase (PCT). Cholesterol and phospholipid concentrations decreased with maturation, phospholipids declining more than cholesterol. Thus, the cholesterol-to-phospholipid molar ratio rose with maturation. A similar decline was seen with PCT activity. Glucocorticoid treatment had no effect on cholesterol, phospholipids, or PCT. However, the maturational increase in the cholesterol-to-phospholipid molar ratio was blunted by glucocorticoid administration in the weanling and adult rats.