Severe iron deficiency affects lipid metabolism. To investigate whether moderate iron depletion also alters lipid variables-including lipid levels in serum and liver, hepatic lipogenesis, and fatty acid composition indicative of an impaired desaturation-we carried out experiments with rats fed 9, 13, and 18 mg iron/kg diet over a total of 5 wk. The study also included three pair-fed control groups and an ad libitum control group, fed with 50 mg iron/kg diet. The iron-depleted rats were classified as iron-deficient on the basis of reduced serum iron, hemoglobin concentration, and hematocrit. All moderately iron-deficient rats had significantly lower cholesterol concentrations in liver and serum lipoproteins than their pair-fed controls. Rats with the lowest dietary iron supply had higher concentrations of hepatic phosphatidylcholine (PC) and phosphatidylethanolamine (PE), lower activities of glucose-6-phosphate dehydrogenase, malic enzyme and fatty acid synthase, and higher triacylglycerol concentrations in serum lipoproteins than the corresponding pair-fed control rats. Moderate iron deficiency also depressed the serum phospholipid level. Moreover, several consistent significant differences in fatty acid composition of hepatic PC and PE occurred within moderate iron deficiency, which indicate impaired desaturation by delta-9 and delta-6 desaturases of saturated and essential fatty acids. We conclude that lipid variables, including cholesterol in liver and serum lipoproteins as well as fatty acid desaturation, reflect the gradations of iron status best and can be used as an indicator of the degree of moderate iron deficiency.