Cholesterol may order or disorder phospholipids. The physiological contribution of cholesterol to the structural order of lens membrane lipids was determined. Cholesterol and phospholipid from bovine lens nuclear and cortical tissue were separated by thin layer chromatography. The effect of cholesterol upon the trans to gauche transition of the hydrocarbon chains was assessed by measuring CH2 infrared stretching band frequencies as cholesterol was added back to the phospholipids. Although the relative cholesterol level of nuclear lipid was much higher than that of the cortex (59 vs. 36 mol%, respectively), the structural order of unfractionated nuclear and cortical lipids were similar at physiological temperature. Cholesterol added to lipids devoid of cholesterol produced a sharp biphasic effect on the structural order of nuclear lipids, increasing the trans conformation from 56% to 0 mol% cholesterol to 74% at 18% cholesterol to 41% trans at 59 mol% cholesterol. Cholesterol addition produced a shallow biphasic change in the percentage trans conformation of cortical lipids. Maximum order (about 40% trans conformation) was seen at a cholesterol level equal to that of intact cortical lipid (36 mol%). The physiological role of cholesterol is to increase the structural order of cortical membrane lipid and decrease order in nuclear lipid. The net result is a similarity in the structural order of cortical and nuclear membrane. We suggest that the different response of cortical and nuclear lipids to added cholesterol is linked to differences in the phospholipid composition between these two lens regions. In the absence of cholesterol, nuclear phospholipids are much more highly ordered than those of the cortex.