Three experimental diets with varied n-6-to-n-3 fatty acid ratios (120, 40 and 8) were prepared by a suitable blending of safflower oil containing 72.5% linoleic (18:2 n-6) acid and non-detectable levels of alpha-linolenic (18:3 n-3) acid, and soybean oil having 56.1% linoleic (18:2 n-6) acid and 7.9% alpha-linolenic (18:3 n-3) acid. These diets were fed to weanling female Wistar/NIN (inbred) rats for 16 wk to assess the impact of altered dietary n-6-to-n-3 fatty acid ratio on erythrocyte membrane (EMS) cholesterol, phospholipids, fatty acid composition and activities of membrane-bound enzymes such as Na+,K+-ATPase, Ca2+, Mg2+-ATPase and acetylcholinesterase. Activities of total and ouabain-sensitive-ATPases were significantly higher in the erythrocyte membranes of rats fed diets with a n-6-to-n-3 fatty acid ratio of 40 compared to other groups, whereas the erythrocyte membrane-bound acetylcholinesterase was significantly different among the three groups. The highest and lowest activities for this enzyme were observed in the dietary groups with n-6-to-n-3 fatty acid ratios of 8 and 40 respectively. However, the EMS of rats fed diets with a n-6-to-n-3 fatty acid ratio of 40 alone had significantly higher Ca2+,Mg2+-ATPase compared to those of other two groups. Significant increases were observed in absolute amounts of cholesterol, phospholipids and molar ratio of cholesterol to phospholipids in the EMS of rats fed a diet with a very high 18:2 n-6-to-18:3 n-3 fatty acid ratio (120) as compared to those from the dietary group with 18:2 n-6-to-18:3 n-3 fatty acid ratio (40), which had the lowest levels of cholesterol, phospholipids and cholesterol-to-phospholipid molar ratio. On the other hand, the EMS from rats fed a diet with a very low n-6-to-n-3 fatty acid ratio (8) had significantly lower cholesterol and higher proportions of stearic (18:0), oleic (18:1 n-9), eicosapentaenoic (20:5 n-3), and docosahexaenoic acids, and a higher ratio of docosahexaenoic (22:6 n-3) acid-to-a-linoleic (18:3 n-3) acid compared to the EMS from a very high n-6-to-n-3 fatty acid ratio of 120. Although these changes in EM fatty acid profiles were expected of the respective dietary regimens, the observed changes in the activities of membrane-bound enzymes could have resulted from their interaction with membrane cholesterol, phospholipids and fatty acyl chains.