Adipose tissue is a major site of cholesterol storage. In an attempt to define mechanisms controlling this process, a variety of nutritional and metabolic alterations were employed and their effects on adipose tissue cholesterol levels were determined by direct chemical analysis. When rats were raised on Purina chow, a linear increase in the cholesterol/DNA ratio in relation to animal weight (from 120 g [5-6 wk] to 700 g [2 yr]) occurred. The rate of cholesterol accumulation was related to the dietary cholesterol load. Cholesterol accumulation by adipose tissue also occurred in rats raised on a cholesterol-free diet and reached levels exceeding those observed in animals fed on a diet containing 0.05 or 0.1% (w/w) cholesterol. In rats maintained on semisynthetic diets containing 0 to 5% (w/w) cholesterol, the serum cholesterol concentration was inversely related to the dietary concentration, suggesting that feedback inhibition of cholesterol formation may be an important determinant of serum cholesterol levels in this species. Early dietary alterations affected adipose tissue levels later in life. Net cholesterol mobilization from adipose tissue also occurred after acute starvation. Comparison of obese mice with nonobese littermate controls showed that the size of the adipose cholesterol pool was proportional to the degree of adipocity because the amount of cholesterol stored per unit glyceride mass was identical. Adipose tissue cholesterol was not affected by animal sex. Thus, adipose tissue cholesterol levels were dependent on animal age, dietary cholesterol load, early nutritional deprivations, and the size of the adipose organ itself.