The effects of sex, dietary fat levels, and ethanol ingestion on rat liver mitochondrial lipids have been studied. Two groups of male animals were fed either a low‐fat diet for about 76 days or a high‐fat diet for about 52 days, and two groups of female animals were fed the same low‐fat diet for about 50 days or the high‐fat diet for about 37 days. Ethanol was substituted isocalorically for carbohydrate and amounted to 36% of total calories. The total as well as individual concentrations of fatty acids, phospholipids, and neutral lipids were determined in all eight groups of animals. Variable changes were observed in the total fatty acid composition of mitochondria from each of the four groups of animals. After ethanol ingestion, there was a decrease in arachidonate/linoleate ratio in males, while no change was observed in females. Increasing the fat content of the diet decreased this ratio in both controls and experimentals, but it did not alter the effects of ethanol on either sex. Presumably, this was due to the fact that corn oil was the only source of lipid. After ethanol ingestion, the total fatty acid concentration increased in all groups of animals except the males fed the low‐fat diet. A decrease was observed in this group. The same pattern of change was reflected in changes in total phospholipid concentrations. In each case, the majority of the concentration change in total phospholipid could be accounted for by changes in phosphatidylcholine (PC). Measurement of choline oxidase (C.O.) showed that ethanol ingestion increased C.O. activity only in the low‐fat group of males. No change was observed in the other three groups. Chronic ethanol ingestion is known to increase the methylation of phosphatidylethanolamine (PE); therefore, in order to decrease PC, the increase in C.O. in the low‐fat males must have been of sufficient magnitude to offset the increase in PE methylation. Increasing the fat content of the diet offset the effect of ethanol on C.O. in males. Neither ethanol nor fat exerted much effect on C.O. in females. These results emphasize the importance of dietary levels of fat as well as sex in the study of liver mitochondria structure and function in relation to ethanol metabolism.