Male rats were fed diets containing olive (00) or evening primrose (EPO) oil (10%w/w), with or without added cholesterol (1%w/w). After 6-week feeding, the lipid and fatty acid compositions, fluidity, and fatty acid desaturating and cholesterol biosynthesis/esterification related enzymes of liver microsomes were determined. Both the OO and EPO diets, without added cholesterol, increased the contents of oleic and arachidonic acids, respectively, of rat liver microsomes. The results were consistent with the increases in Δ⁹ and Δ⁶ desaturation of n-6 essential fatty acids and the lower microviscosity in the EPO group. Dietary cholesterol led to an increase in the cholesterol content of liver microsomes as well as that of phosphatidylcholine (PC). The cholesterol/phospholipid and PC/PE (phosphatidylethanolamine) ratios were also elevated. Fatty acid composition changes were expressed as the accumulation of monounsaturated fatty acids, with accompanying milder depletion of saturated fatty acids in rat liver microsomes. In addition, the arachidonic acid content was lowered, with a concomitant increase in linoleic acid, which led to a significant decrease in the 20:4/18:2 ratio in comparison to in animals fed the cholesterol-free diets. Cholesterol feeding also increased Δ⁹ desaturase activity as well as membrane microviscosity, whereas it decreased Δ⁶ and Δ⁵ desaturase activities. There was a very strong correlation between fluidity and the unsaturation index reduction in the membrane. Furthermore, the activity of hydroxymethylglutaryl-CoA reductase increased and the activity of acyl-CoA: cholesterol acyltransferase decreased in liver microsomes from both cholesterol-fed groups. This seems to indicate that the responses of these enzyme activities to the different diets take place in order to maintain the homeostasis of membranes.